JP2015517526A - Effective vaccination before weaning for porcine reproductive and respiratory syndrome (PRRS) virus - Google Patents

Abstract

The present invention provides an isolated polynucleotide molecule comprising a DNA sequence that encodes an infectious RNA sequence encoding a genetically engineered North American PRRS virus, a method of making it, and related polypeptides, polynucleotides, and various components To do. Also provided are vaccines comprising this genetically modified virus and polynucleotide, and diagnostic kits for distinguishing between naturally infected animals and vaccinated animals. [Selection] Figure 5

Description

The present invention relates to the field of animal health, infectious cDNA clones of positive polarity RNA viruses, new RNA viruses and modified live forms thereof, and construction of vaccines, in particular such c
The subject is a pig vaccine using a DNA clone. More specifically, the present invention also provides safe and early vaccination of pre-weaning piglets, including shortly after birth (ie, less than 1 day of age) to 2 weeks of age, always selective, Valence mixed swine vaccine, eg divalent PRRSV /
Mycoplasma hyopneumoniae (M.hyo) vaccine, bivalent PRRSV / pig circovirus type 2 (PCV2) vaccine, and trivalent PRRSV / M. Inoculated as a combination with a hyo / PCV2 vaccine or simply as a monovalent PRRSV vaccine. Early vaccination against PRRS under such circumstances provides early development of protective immunity, which is within about 14 days after vaccination, ie 15 days after vaccination at 1 day of age, 7 days of age. Occurs within about 28 days after vaccination at 21 days, 14 days of age. This specification provides a number of constructs of the “P129 strain” of North American PRRS virus (No. PCT / IB).
2011/055003 and US 6,500,662), including the early and safe use described above, which is highly effective as a vaccine, but early onset of such protective immunity (ie early in the first day of life) About 2 weeks after vaccination given to other North American and European P
RRS strains such as US 5,476,778, US 5,846,805, US 6
, 380,376, US Pat. No. 6,982,160 and US Pat. No. 6,197,310.

Porcine reproductive and respiratory syndrome (PRRS) is characterized by miscarriages, stillbirth, and reproductive problems in other sows and gilts, and respiratory disease in young pigs. The pathogen is the PRRS virus (PRRSV), a member of the Arteriviridae and Nidoviridae. The order Nidovirus is an enveloped virus with a genome composed of a single strand of positive polarity RNA. The genomic RNA of a positive strand RNA virus plays a dual role in both conservation and expression of genetic information. Replication or transcription in Nidovirus does not include DNA. Nonstructural proteins are converted directly from Nidovirus genomic RNA as large polyproteins, which are then cleaved by viral proteases to become humble and functional proteins. A set of 3 ′ co-terminal nested sets of subgenomic RNA (sgRNA) is synthesized from the genome and used as messenger RNA for the conversion of structural proteins. Regeneration of Nidovirus genomic RNA is thus possible with genomic replication and sgRNA
It is a composite process of synthesis.

In the late 1980s, two different genotypes of the virus appeared almost simultaneously in North America and the other in Europe. The PRRS virus is now endemic in almost all pig-producing countries and is considered to be one of the economically significant diseases affecting the global pig industry. In addition, highly toxic genotypes have been removed in China and neighboring countries, and such genotypes are generally associated with North American genotypes.

Despite significant advances in understanding the biology of PRRSV, virus control remains difficult. Vaccination of outdoor animals has generally proved ineffective. P
RRS is generally re-expressed in the vaccinated herd and PR on most farms
RSV vaccination campaigns cannot ultimately control the disease.

Without being limited to theory, infection of pigs with wild-type PRRSV, or vaccination with live attenuated forms of this pathogen, unfortunately only induces abundant production of non-neutralizing antibodies. During this time interval, for example, a limited amount of interferon (IFN) -γ (only secreting cells are produced. Therefore, PRRSV is essentially always abundant humoral (antibody based) immunity, and Variety of limited but potentially protective T helper (Th) 1 like IF
It is thought to stimulate an unbalanced immune response, distinguished by an N-γ response. One feature of PRRSV infection that appears to be most involved in the progression of adaptive immunity is the lack of a sufficient innate immune response. Usually, virus-infected cells are type I interferon “IFN” (IF
Secretes N-α and IFN-β) and protects adjacent cells from infection. Furthermore, the released type I IFN interacts with a subset of naïve T cells, facilitating conversion to virus-specific type II IFN (IFN-γ) secreting cells. In contrast, the porcine IFN-α response to PRRSV exposure is virtually non-existent. Such ineffective stimulation by IFN-α producing pathogens would be expected to have a significant impact on the essence of the host's adaptive immune response, as IFN-α increases IFN-γ gene expression. Thus, the original cytokine controls the dominant pathway and promotes the development of adaptive immunity, ie T cell mediated IFN-γ response and maximal antiviral immune defense.

In this regard, a possible link between innate and adaptive immunity in viral infection is the specialization of dendritic cells that play an important role in the polarity formation of T-cell function that can produce large amounts of type I interferon. It is clear that this is caused by the mold. Specifically, plasmacytoid dendritic cells (PDCs), which are rare but excellent dendritic cell types and also known as natural IFN-α / β-producing cells, convert naive T cells into IFN-γ secreting cells. The ability to differentiate into plays an important role in antiviral immunity. Although rare, PDC are very potent IFN-α producing cells, each capable of producing 3-10 pg IFN-α in response to virus. In contrast, monocytes produce 5 to 10 times less IFN-α per cell. The phenotype and some biological properties of porcine PDC have been described (Summerfield et al., 2003, Immunology 110: 440). Recent studies have shown that PRRSV does not stimulate IFN-α secretion in porcine PDC (Calzada et al., 2010, Veterinary Immunology and Immunopathology 135: 20).

This fact suggests that IFN-α added from the outside at the time of vaccination
The observation of improving the intensity of the N-γ reaction (WA Meier et al., Vet.
Immunol. Immunopath. 102, pp 299-314, 2004) highlights the important role that IFN-α plays during infection of this virus in swine. Given the apparently important role of IFN-α in the development of protective immunity, different PR
It is important to determine the ability of RS virus stock to stimulate and / or inhibit IFN-α production. Therefore, there is an urgent need for new and improved modified live vaccines to protect against PRRS. New infectious cDNA clone, pCMV-S-
Viruses generated from P129-PK and others are wild type P129 virus or 2
It is clear that it has a different phenotype from some commercial modified live PRRS vaccines. Without being limited to theory, the present invention provides a new and effective generation of vaccines and PRRS vaccines that promote immune responses to cell-based viruses.

In a first embodiment, the present invention provides a DNA that encodes an infectious RNA molecule that encodes a PRRS virus that is genetically modified and induces an effective immunoprotective response against the PRRS virus in pigs as a vaccine. A separate polynucleotide molecule comprising the sequence is provided. In certain embodiments, the invention provides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, or at least 70% identity thereto, preferably 8
0% identity, more preferably 85%, 90%, 95%, 96%, 97%, 98%
Alternatively, the DNA sequences described herein are provided that contain sequences with 99% identity.

In certain embodiments, the present invention provides a plasmid comprising the isolated polynucleotide molecule described herein and a promoter gene capable of transcribing the polynucleotide molecule in a suitable host cell. In another embodiment, the North American or Chinese PRRS coding sequences of the plasmids described herein further encode one or more detectable heteroantigenic epitopes. The present invention provides nucleic acid introduced host cells having the plasmids described herein.

In another aspect, the invention provides a vaccine that protects pigs against infection with PRRS virus. The vaccine can include a North American or Chinese PRRS virus encoded by an infectious RNA molecule, each of which is encoded by an isolated polynucleotide molecule described herein. In yet other aspects, the vaccine comprises a viral vector comprising a polynucleotide described herein. The vaccine set described herein may optionally include a vaccine carrier that is acceptable for use on livestock. In one important aspect, the vaccine has a reduced interferon-α inhibitory effect compared to wild-type P129PRRS virus (see ATCC 203488, 203489, US Pat. No. 6,500,662).

In certain embodiments, the present invention distinguishes between pigs naturally infected with a field strain of PRRS virus and pigs vaccinated with the modified live vaccine set described herein (
So-called DIVA testing) Diagnostic kits containing polynucleotide molecules are provided.

In another embodiment, the present invention provides a method for treating pigs from infection with a PRRS virus strain, comprising administering to an animal an immunogenically protective amount of a vaccine as claimed herein. Provide a way to protect.

A further and preferred embodiment of the present invention provides for isolated porcine reproductive and respiratory syndrome virus (
PRRS), or a polynucleotide sequence code therefor, wherein the protein encoded by ORF1a is selected from the group consisting of any of the following amino acid sequences, and the underlined residues appear to be new. AMA N VYD (SEQ ID NO: 9), IGH N AVM (SEQ ID NO: 12), TVP D GNC (SEQ ID NO: 15), CWW Y LFD (SEQ ID NO: 18), H
GV H GKY (SEQ ID NO: 21), AAK V DQY (SEQ ID NO: 24), PSA T DTS (SEQ ID NO: 27), LNS L LSK (SEQ ID NO: 30), APM C QDE (SEQ ID NO: 33), C
AP T GMD (SEQ ID NO: 36), PKV A KVS (SEQ ID NO: 39), AGE I VGV (SEQ ID NO: 42), ADF N PEK (SEQ ID NO: 45), and QTP I LGR (SEQ ID NO: 48)
). In a further preferred embodiment of the present invention, the present invention has the sequences identified above in the protein encoded from ORF1a and from any combination of these identified sequences (2, 3, 4, up to Supply North American or Chinese PRRS separations, including 17).

The invention further provides isolated porcine reproductive and respiratory syndrome virus (PRRS), wherein the protein encoded by ORF1a is selected from the group consisting of amino acid sequences comprising any of the following: A N V (see SEQ ID NO: 9), H N A (see SEQ ID NO: 12), P D G (see SEQ ID NO: 15), W Y L (see SEQ ID NO: 18), V H G (see SEQ ID NO: 21), K V D (see SEQ ID NO: 24), (see SEQ ID NO: 27) A T D, (see SEQ ID NO: 30) S L L, (see SEQ ID NO: 33) M C Q, P T G ( see SEQ ID NO: 36), V a K (see SEQ ID NO: 39), E I V (see SEQ ID NO: 42), F N P (see SEQ ID NO: 45), and P I L (see SEQ ID NO: 48), any combination of these identified sequences ( 2, 3, 4, and up to 17).

In a further preferred embodiment, the invention provides isolated North American or Chinese PRRS, where any other specific nucleotide or amino acid sequence position at any point where the polynucleotide encodes a virus or protein encoded therefrom. Regardless of the identity of the ORF1a viral protein,
(A) amino acid N in amino acid sequence A N V of any of the specific amino acids in the following specific sequences (see SEQ ID NO: 9);
(See SEQ ID NO: 12) amino acid N in the amino acid sequence H N A,
(See SEQ ID NO: 15) amino acid D in the amino acid sequence P D G,
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18);
Amino acid H in the amino acid sequence V H G (see SEQ ID NO: 21),
Amino acid V in the amino acid sequence K V D (see SEQ ID NO: 24),
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27);
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30);
Amino acid C in amino acid sequence M C Q (see SEQ ID NO: 33);
(See SEQ ID NO: 36) amino acid T in the amino acid sequence P T G,
Amino acid A in amino acid sequence V A K (see SEQ ID NO: 39),
(See SEQ ID NO: 42) amino acid I in the amino acid sequence E I V,
Amino acid N in amino acid sequence F N P (see SEQ ID NO: 45) and amino acid I in amino acid sequence P I L (see SEQ ID NO: 48), any combination of these identified sequences (2, 3, 4, up to 17) Including, or
(B) matches the above three residue sequences, taking into account that the other specified three residue amino acid sequences may show one or two additional amino sequence changes but are still considered to match the specified sequences Including the specific underlined single amino acid in the specific 3-residue ORF1a peptide sequence of any other North American or Chinese PRRS virus. For purposes of this embodiment of the invention, “match” refers to the related sequence as described in Henikoff et al. Proc Natl. Acad. Sci. USA, 89, pp. 10915-10919, 1992, which means that it can be optimally aligned using the BLOSUM algorithm.

In a further preferred embodiment of the invention, an isolated porcine reproductive and respiratory syndrome virus (PRRS) is provided, wherein the protein encoded by ORF1a is altered (a), (b), (c) and ( d) having an amino acid sequence having one or more of the following, each change being defined as follows:
Change (a),
Amino acid N in amino acid sequence A NV (see SEQ ID NO: 9)
Amino acids in the amino acid sequence H N A N (see SEQ ID NO: 12)
Amino D in the amino acid sequence P D G (see SEQ ID NO: 15)
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18)
Amino acid H in the amino acid sequence V H G (see SEQ ID NO: 21), or any subset of changes (a);
Change (b),
Amino acid V in amino acid sequence K V D (see SEQ ID NO: 24)
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27)
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30)
Amino acid C in amino acid sequence M C Q (see SEQ ID NO: 33), or any subset of changes (b);
Change (c),
Amino T in the amino acid sequence P T G (see SEQ ID NO: 36)
Amino acid A in amino acid sequence V A K (see SEQ ID NO: 39), or any subset of changes (c), as well as changes (d),
(See SEQ ID NO: 42) amino acid I in the amino acid sequence E I V,
Amino acid N in amino acid sequence F NP (see SEQ ID NO: 45),
And amino acid I in amino acid sequence P I L (see SEQ ID NO: 20), or any subset thereof (d).

  The PRRS virus is further identified by two or more of the five amino acid sequences identified in change (a) and / or two or more of the four amino acid sequences identified in change (b) and / or in change (c) 2 It may comprise two amino acid sequences and / or two or more of the three amino acid sequences identified in change (d).

The present invention also provides a plasmid capable of directly introducing nucleic acid into an appropriate host cell and expressing porcine reproductive and respiratory syndrome virus (PRRS) from the appropriate host cell into which the nucleic acid has been introduced. a) a DNA sequence encoding an infectious RNA molecule encoding a PRRS virus, and (b) a promoter capable of transcribing said infectious RNA molecule, wherein the protein encoded by ORF1a of said virus is 1) amino acid N in amino acid sequence A N V (see SEQ ID NO: 9),
(See SEQ ID NO: 12) amino acid N in the amino acid sequence H N A,
(See SEQ ID NO: 15) amino acid D in the amino acid sequence P D G,
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18);
Amino acid H in amino acid sequence V H G (see SEQ ID NO: 21), or any subset thereof, and / or (2) amino acid V in amino acid sequence K V D (see SEQ ID NO: 24),
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27);
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30);
(See SEQ ID NO: 33) amino acid C in the amino acid sequence M C Q, or any subset thereof, and / or (3) (see SEQ ID NO: 36) amino acid T in the amino acid sequence P T G,
Amino acid A in amino acid sequence V A K (see SEQ ID NO: 39), or any subset thereof, and / or (4) amino acid I in amino acid sequence E I V (see SEQ ID NO: 42),
Amino acid N in amino acid sequence F NP (see SEQ ID NO: 45),
And amino acid sequence comprising amino acid I in amino acid sequence P I L (see SEQ ID NO: 48), or any subset thereof.

  It will be appreciated that ORF1a encodes a polyprotein with pro-asease function and ORF1b encodes a polyprotein with replicase (RNA polymerase) and helicase function. Additional information regarding the function of proteins encoded from various PRRS ORFs (translation regions) can be found, for example, in US Pat. No. 7,132,106. See also US Pat. No. 7,544,362 for the function of ORF7 and other translation regions. As will be appreciated by those skilled in the art, ORF1-encoding proteins are expected to have additional functions, including known and unknown, and novel amino acid changes that are useful in the practice of the present invention are ORF-1 It is not limited to those that affect any one of the specific functions of the encoded protein.

  In a further preferred embodiment, said plasmid comprises a promoter, i.e. a eukaryotic promoter capable of allowing DNA initiation into the target eukaryotic cell, or a prokaryotic or eukaryotic capable of inducing in vitro transcription of the plasmid. It is a phage promoter. The present invention also provides a method for producing PRRS virus, which method comprises introducing a suitable host cell with a suitable plasmid and nucleic acid and obtaining a PRRS virus produced by the nucleic acid introduced cell.

Accordingly, in certain preferred embodiments, the invention provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, wherein the DNA sequence is from the group consisting of Selected,
(A) SEQ ID NO: 6,
(B) a sequence having at least 85% identity with the DNA sequence of (a), wherein the protein encoded by ORF1a is:
Amino acid N in amino acid sequence A N V from group (b) (1) (see SEQ ID NO: 9);
(See SEQ ID NO: 12) amino acid N in the amino acid sequence H N A,
(See SEQ ID NO: 15) amino acid D in the amino acid sequence P D G,
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18);
Amino acid H in amino acid sequence V H G (see SEQ ID NO: 21), or any subset thereof, and / or amino acid V in amino acid sequence K V D from group (b) (2) (see SEQ ID NO: 24),
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27);
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30);
Amino acids in the amino acid sequence M C Q C (see SEQ ID NO: 36) amino acid T in (see SEQ ID NO: 33), or the amino acid sequence from any subset thereof, and / or group (b) (3) P T G,
Amino acid sequence V A acid in K A (see SEQ ID NO: 39), or any subset thereof, and / or group (b) (see SEQ ID NO: 42) amino acid I in the amino acid sequence E I V (4),
Amino acid N in amino acid sequence F NP (see SEQ ID NO: 45),
And a sequence comprising an amino acid sequence comprising amino acid I in amino acid sequence P I L (see SEQ ID NO: 20), or any subset thereof, and (c) 0.5M NaHPo4, 7% SDS, 1 mM EDTA Complement to the DNA sequence of (a) or (b) under high stringency conditions including hybridization to filter-bound DNA at 65 ° C. in solution and washing at 68 ° C. in 0.1 SSC / 0/1% SDS A DNA sequence that hybridizes.

  The present invention also provides a host cell nucleic acid introduced into a polynucleotide molecule and a vaccine that protects pigs from PRRS virus infection, the vaccine being encoded by (a) such a polynucleotide molecule as described above, A recombinant North American PRRS virus, or (b) the infectious molecule, or (c) the polynucleotide molecule in the plasmid form, or (d) a viral vector comprising the polynucleotide molecule, wherein the PRRS virus is immune to infection An effective immune protection reaction against PRRS virus infection in an amount effective for protective production and a suitable carrier for use in livestock can be induced.

The present invention also provides RNA polynucleotide sequences that are consistent with the following (ie, having a complementary base coding sequence).
(A) the DNA sequence of SEQ ID NO: 6,
(B) a DNA sequence having at least 85% identity with the DNA sequence of (a), and the protein encoded by ORF1a has an amino acid sequence comprising any of the following and any combination of the following: Amino acid N in the DNA sequence amino acid sequence A N V (see SEQ ID NO: 9),
(See SEQ ID NO: 12) amino acid N in the amino acid sequence H N A,
(See SEQ ID NO: 15) amino acid D in the amino acid sequence P D G,
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18);
Amino acid H in the amino acid sequence V H G (see SEQ ID NO: 21),
Amino acid V in the amino acid sequence K V D (see SEQ ID NO: 24),
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27);
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30);
Amino acid C in amino acid sequence M C Q (see SEQ ID NO: 33);
(See SEQ ID NO: 36) amino acid T in the amino acid sequence P T G,
Amino acid A in amino acid sequence V A K (see SEQ ID NO: 39),
(See amino acid I (SEQ ID NO: 42 in the amino acid sequence E I V),
Amino acid N in amino acid sequence F NP (see SEQ ID NO: 45),
And amino acid I in amino acid sequence P I L (see SEQ ID NO: 20), or (c) hybridization to filter-bound DNA at 65 ° C. in a solution of 0.5 M NaHPo4, 7% SDS, 1 mM EDTA and A DNA sequence that hybridizes to the complement of the DNA sequence of (a) or (b) under high stringency conditions including washing at 68 ° C. in 0.1 SSC / 0/1% SDS.

  Accordingly, the present invention also provides a diagnostic kit comprising a polynucleotide molecule that distinguishes between pigs naturally infected with a field strain of PRRS virus and pigs vaccinated with the vaccines described herein. ) Preferably demonstrates an attenuated interferon-α inhibitory effect compared to wild type P129PRRS virus (SEQ ID NO: 5).

BRIEF DESCRIPTION OF THE TABLES AND FIGURES Table 1 shows infectious cDNA clones and their corresponding viruses induced by introduction of nucleic acids into PK-9 cells.
Table 2 shows the interferon-α inhibitory effect of wild-type PRRS virus and derivatives adapted for growth in cell culture.
Table 3 shows the interferon-α inhibitory effects of genetically engineered derivatives adapted for growth in wild type PRRS virus P129 and CD163 expressing PK-9 cells.
Table 4 compares the wild-type P129 virus and the PRRS Ingelvac vaccine,
The reduced interferon-alpha inhibitory effect of P129-PK-FL and P129-PK-d43 / 44 viruses is shown.
Table 5 shows the study concept for assessing the safety and efficacy of vaccine viruses.
Table 6 shows all nucleotide differences and resulting amino acid differences between passage 0 and passage P129-PK-FL by gene position.
Table 7 shows passage 0 and passage 17 P129-PK-FL by viral protein.
The summary of nucleotide and amino acid differences between and is shown.
Table 8 shows the infectious cDNA clones pCMV-S-P129 and p by gene location.
Figure 5 shows all nucleotide differences and the resulting amino acid differences between the PRRSV genes found in CMV-S-P129-PK17-FL.
Tables 9 and 10 show the amino acid changes that contribute to the phenotype (SEQ ID NO: 6) of passage 52 virus.
Table 11 shows the number of pigs with clinical symptoms after inoculation with modified live PRRSV vaccine at 1 day of age.
Table 12 shows the mean serum titer after inoculation with modified live PRRSV vaccine at 1 day of age.
Table 13 shows the percentage of lung lesions after challenge of 7 week old pigs previously vaccinated with a modified live PRRSV vaccine at 1 day of age.
Table 14 shows the percentage of lung lesions after challenge of 18 week old pigs previously vaccinated with a modified live PRRSV vaccine at 1 day of age.
Table 15 shows the percentage of lung lesions after challenge of 26 week old pigs previously vaccinated with a modified live PRRSV vaccine at 1 day of age.
Table 16 shows the percentage of lung lesions after challenge of 5 week old piglets previously vaccinated with live modified PRRSV vaccine.

FIG. 1 illustrates rectal temperature after vaccination. FIG. 2 illustrates rectal temperature after challenge with toxic PRRSV NADC20. FIG. 3 illustrates the body weight after vaccination and after antigen administration. FIG. 4 illustrates the data after challenge of the proportion of lungs with PRRS lesions. FIG. 5 illustrates the lung assessment score (LAS) for the severity of the observed lesions after challenge. FIG. 6 is a histogram illustrating anti-PRRSV antibody levels (ELISA S / P ratio) in sera after vaccination and challenge. FIG. 7 is a graphical representation of viral load in serum (log TCID 50 / ml in PAM cells) after antigen administration. FIG. 8 is a graphical representation of the method used to obtain a vaccine comprising SEQ ID NO: 1 to SEQ ID NO: 6 disclosed herein.

Brief Description of Major Sequences SEQ ID NO: 1 provides the complete genome of P129-PK-FL passage 17.

  SEQ ID NO: 2 provides the complete genome of P129-PK-d43 / 44 passage 17.

  SEQ ID NO: 3 provides the complete genome of P129-PK-FL passage 24.

  SEQ ID NO: 4 provides the complete genome of P129-PK-d43 / 44 passage 34.

  SEQ ID NO: 5 provides the complete genome of P129 passage 0.

  SEQ ID NO: 6 provides the complete genome of P129 passage 52.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include references to the plural unless the context clearly indicates otherwise. Thus, for example, reference to “the present method” includes one or more of the methods and / or steps described herein and will be apparent to those of ordinary skill in the art upon reading this disclosure and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are now described.

The practice of the present invention, unless specified to the contrary, virology, immunology, microbiology,
Many are described below for purposes of illustration, using conventional methods within the skill of a person skilled in the art of molecular biology and genetic engineering. Such techniques are explained fully in the literature. See, for example, Sambrook, et al. Molecular Cloni
ng: A Laboratory Manual (2nd Edition, 1989)
Maniatis et al. Molecular Cloning: A Labo
ratato Manual (1982); DNA Cloning: A Practi
cal Approach, vol. I & II (D. Glover, ed.);
onotide Synthesis (N. Gait, ed., 1984); N
ucleic Acid Hybridization (B. Hames & S. Higg
ins, eds. , 1985); TRANSCRIPTION AND TRANSLA
tion (B. Hames & S. Higgins, eds., 1984);
Cell Culture (R. Freshney, ed., 1986); Perba
l, A Practical Guide to Molecular Cloning
(1984).

“North American PRRS virus” refers to any PRRS virus having genetic characteristics associated with North American PRRS virus isolates, such as P first isolated in the United States in the early 1990s.
RRS viruses (eg, Collins, JE, et al., 1992, JV)
et. Diagn. Invest. 4: 117-126); North America
n PRRS virus isolate MN-1b (Kwang, J. et al
. , 1994, J. et al. Vet. Diagn. Invest. 6: 293-296); the
Quebec LAF-exp91 strain of PRRS (Mardassi
, H .; et al. , 1995, Arch. Virol. 140: 1405-1418)
; And North American PRRS virus isolate VR
2385 (Meng, X.-J et al., 1994, J. Gen. Virol.
75: 1795-1801)), but is not limited thereto. Genetic features refer to genomic nucleotide sequence similarity and amino acid sequence similarity shared with North American PRRS virus strains. Chinese RPPS virus strains generally display about 80-93% nucleotide sequence similarity with North American strains.

“European PRRS virus” means any strain of PRRS virus having genetic characteristics associated with PRRS virus isolated for the first time in Europe around 1991 (eg Wen
svoort, G .; , Et al. 1991, Vet. Q. 13: 121-130). “European PRRS virus” may also mean “Leystad virus” to those skilled in the art.

“Effective immunoprotective response”, “immunoprotection” and similar terms are used for purposes of the present invention to refer to one or more pathogen antigenic epitopes to protect against infection by pathogens in vaccinated animals. Means an immune response directed at For the purposes of the present invention,
Protection from infection by a pathogen is not only complete protection from infection, but also any detectable attenuation of the extent or rate of infection by the pathogen, or vaccinated compared to an unvaccinated infected animal. Also includes any detectable attenuation of the severity of the disease or any symptom or condition resulting from infection with a pathogen in a living animal. An effective immunoprotective response can be induced in animals that have not been previously infected with a pathogen and / or at the time of vaccination. An effective immune protective response can also be induced in animals already infected with pathogens at the time of vaccination.

A recombinant PRRS virus is “attenuated” if it is less toxic than its unmodified parental strain. A strain is “less toxic” when it shows a statistically significant decay in one or more parameters that determine the severity of the disease. Such parameters may include the level of viremia, fever, severity of dyspnea, severity of reproductive symptoms, or the number or severity of lung lesions.

“Host cell capable of supporting PRRS virus replication” means a cell capable of producing infectious PRRS when infected with a virus of the invention. Such cells include monocyte / macrophage lineage porcine cells such as porcine alveolar macrophage cells and derivatives, MA-104 monkey kidney cells and derivatives such as MARC-145 cells, and PRR.
Including the S virus receptor and nucleic acid introduced cells. The term “host cell capable of supporting PRRS virus replication” may also include live porcine cells.

As used herein, “translation region” or “ORF” means the minimal nucleotide sequence required to encode a particular PRRS viral protein without an intervening stop codon.

“Porcine” and “swine” are used interchangeably herein and mean any animal that is a member of the wild boar family, for example, a pig. The term “PRRS virus” as used herein means a strain of either North America or European PRRS virus, unless otherwise specified.

“PRRS” includes disease symptoms in pigs caused by PRRS virus infection. Examples of such symptoms include, but are not limited to, fever, miscarriage in pregnant females, dyspnea, lung lesions, loss of appetite, and young pig mortality. As used herein,
A PRRS virus that "cannot produce PRRS" can infect pigs, but P
It refers to a virus that does not produce disease symptoms commonly associated with RRS infection.

PRRSV “N protein” or “ORF7” as used herein is defined as a polypeptide encoded by ORF7 of both the European and North American genotypes of PRRS virus. Examples of specific isotypes of N proteins currently known include the North American PRRS123 amino acid polypeptide prototype isolate VR2322 reported in Genbank by accession number PRU87392, and Genbank accession number A26843.
The 128-residue N protein of the European prototype PRRS isolate Lelystad reported in.

"PRRSV N protein NLS-1 region" or "PRRSV ORF7 NLS
-1 region "means" pat4 "or" nucl "nuclear localization signal (Nakai &
Kanehisa, 1992; Rowl & Yoo, 2003) containing four consecutive basic amino acids (lysine or arginine) or three basic residues and histidine or proline, located approximately within the first 15 N-terminal residue of the mature N protein To do. As an example, L
The elystad isolate sequence is KKKK and is located at residues 10-13 of the N protein, while the VR2332 NLS-1 region sequence is KRKK and is located at residues 9-12.

"PRRSV N protein NLS-2 region" or "PRRSV ORF7 NLS
-2 region "refers to the second nuclear localization signal within the N protein, one of two forms
Can take one form. In North American PRRS virus, NLS-2 is “pat”
It has a pattern designated as an “8” motif and starts with a proline followed within 3 residues by a 5 residue sequence and contains at least 3 basic residues (K or R) out of 5 (“pat7 ”Or“ nuc2 ”motif is slightly modified by Nakai & Kane
hisa, 1992, Rowland & Yoo, 2003). By way of example, such a sequence is located in N protein residues 41-47 of North American PRRSV isolate VR2332, . . Represented by K. In European PRRS virus, NLS-2 is “pa
A continuous spread of four basic amino acids or three basic residues related to histidine or proline with a “t4” or “nuc1” motif (Nakai & Kane
hisa, 1992; Rowland & Yoo, 2003). European PRRSV isolate Le
NLS-2 of lystad is located at residues 47-50 and has the sequence K. . Represented by K.

“PRRSV N protein NoLS region” or “PRRSV ORF7 NoLS”
“Region” means a nucleolar localization signal having a total length of about 32 amino acids and encompassing the NLS-2 region near its amino terminus. As an example, the VR2332 NoLS region sequence is residues 41-7
2 and the sequence P.I. . . The corresponding Lelystad isolate sequence represented by R (Rowl & Yoo, 2003) is located at residues 42-73 and contains the sequence P.P. . Represented by R.

“Nucleic acid-introduced host cell” means virtually any host cell described in US Pat. No. 5,600,662, and when nucleic acid is introduced into a PRRS virus, RNA is at least a first of the PRRS viral particles. Rounds can be generated. An “infectious DNA molecule” in the present invention encodes an essential element to support replication, transcription and conversion from a suitable host cell to a functional viral particle. Similarly, an “isolated polynucleotide molecule” is
It means a composition of matter comprising a polynucleotide molecule of the present invention, purified to any degree detectable from a naturally occurring state.

  In the present invention, the nucleotide sequence of the second polynucleotide molecule (either RNA or DNA) is “cognate” with the nucleotide sequence of the first polynucleotide molecule, or “ The nucleotide sequence of the second polynucleotide molecule encodes the same polyamino acid as the nucleotide sequence of the first polynucleotide molecule, based on the degeneracy of the genetic code, or the first polynucleotide It means a case where it encodes a polyamino acid sufficiently similar to the polyamino acid encoded by the nucleotide sequence of the molecule and is useful in the practice of the present invention. A cognate polynucleotide sequence also refers to the sense and antisense strands, and in all cases the complement of any such strand. In the present invention, polynucleotide molecules are useful in practicing the present invention, and thus are homologous or identical and detect the presence of a PRRS virus or viral polynucleotide in a bodily fluid or tissue sample of an infected pig, for example, a standard It can be used as a diagnostic probe by hybridization or amplification techniques. Generally, a first polynucleotide molecule has a nucleotide sequence identity when the nucleotide sequence of the second polynucleotide molecule has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the BLASTN algorithm. (National Center for Biotechnology Information, or NCBI, (Thethesda, Maryland, USA) of the United States National Institute of Health). In a specific example of a calculation according to the practice of the present invention, BLASTP 2.2.6 [Tatsusova TA and TL Madden, “BLAST 2 sequences-a new tool for preparing protein and nucleotide sequences.” 1999 1999Ft. 174: 247-250. ]. Briefly, two amino acid sequences are used with “blosum 62” to score a gap opening penalty of 10, a gap extension penalty of 0.1, and a Henikoff and Henikoff matrix to optimize the alignment score. (Proc. Nat. Acad. Sci. USA 89: 10915-10919.1992). Percent identity is then calculated as follows: total number of identical matches × 100/2 the longer gap sequence introduced into the longer sequence to align the two sequences + the length of the number.

  Preferably, the cognate nucleotide sequence has at least about 75% nucleotide sequence identity, more preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% Nucleotide sequence identity. Because the genetic code is degenerate, a cognate nucleotide sequence can contain any number of “silent” base changes, ie, nucleotide changes but base changes that encode the same amino acid.

The cognate nucleotide sequence can further include non-silent mutations, i.e., base substitutions, deletions, or additions, and is at least about 70% identical to the polyamino acid encoded in the first nucleotide sequence or in the practice of the invention. As long as it is beneficial, it results in amino acid differences in the encoded polyamino acids. In this regard, certain conservative amino acid substitutions that are generally recognized not to inactivate overall protein function, eg, amino acids (as well as vice versa), lysine, aridinine and histidine as having a positive charge; And (1) alanine and serine, (2) asparagine as amino acids (and vice versa), aspartic acid and glutamic acid; , Glutamine and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine and tyrosine, ( 8) Serine and threonine, 9) tryptophan and tyrosine, (10) and such as tyrosine, tryptophan and phenylalanine may be made. Amino acids can be classified by physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized by those skilled in the art as a substitution of one amino acid for another with similar properties. Exemplary conservative substitutions can be found in International Publication No. WO 97/09433, published March 13, 1997, page 10, (International Application No. PCT / GB96 / 02197 filed September 6, 1996). Or, conservative amino acids can be obtained from Lehninger (Biochemistry, Second Edition;
Worth Publishers, Inc. NY: NY (1975), pp. 71-7
Can be classified as described in 7). Further suitable conservative changes and their applications are described below.

Cognate nucleotide sequences can be determined by comparison of nucleotide sequences, eg, using BLASTN as described above. Alternatively, cognate nucleotide sequences can be determined by hybridization under selective conditions. For example, hybridization to filter bound DNA at 65 ° C. in a solution of 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA under moderate stringency conditions, and 0.2 × SSC Wash at 42 ° C. in 0.1% SDS (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp. 6.0.3-6.4.10) or otherwise When hybridized to the complement of SEQ ID NO: 1 under conditions that result in hybridization of a sequence encoding a PRRS virus as defined below, the nucleotide sequence of the second polynucleotide molecule is SEQ ID NO: 1 (or any other specific Polynucleoti Array) and is a cognate. The modification of hybridization conditions can be determined empirically or can be calculated accurately based on the length and proportion of guanosine / cytosine (GC) base pairing of the probe. Hybridization conditions are described in Sambrook, et al. (Eds.), Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. It can be calculated as described in 9.47 to 9.51.

In another embodiment, the complement of SEQ ID NO: 1 is subjected to high stringency conditions at 65 ° C. in a solution of 0.5 M NaHPO ₄ , 7% SDS, 1 mM EDTA, as is well known to those skilled in the art. The second nucleotide sequence is SEQ ID NO: 1 (or any other of the present invention), when hybridized to the filter bound DNA and washed at 68 ° C. in 0.1 × SSC / 0.1% SDS. (Ausebel et al. Current Protocols in Molecular Biology, Green Publishing and Wiley Interscience, New York, 1989).

  Furthermore, isolated polynucleotide molecules and isolated RNA molecules in the present invention include both synthetic molecules and molecules obtained by recombinant techniques such as in vitro cloning and transcription.

Polynucleotide molecules can be genetically mutated using recombinant techniques well known to those skilled in the art, and can be site-directed mutagenesis or random mutagenesis, eg, to chemical mutagens or radiation as is well known to those skilled in the art. Including exposure. Mutations can be performed by standard methods well known to those skilled in the art, such as infectious copies as described (eg, Müllenberg et al., Ad.
v. Exp. Med. Biol. , 1998, 440: 199-206) (for example, Sambrook et al. (1989) Molecular C).
lonning: A Laboratory Manual, 2nd Ed. , Cold
Spring Harbor Laboratory Press, Cold Spri
ng Harbor, N .; Y. See).

Accordingly, the present invention further provides a method for producing a recombinant North American PRRS virus,
The method comprises a D encoding an infectious RNA molecule encoding a PRRS virus as described above.
Mutating the NA sequence and using a suitable expression system, recombinant PRRS
Expressing the virus. The recombinant PRRS virus can be expressed from isolated polynucleotide molecules using an appropriate expression system well known to those skilled in the art, examples of which are described in this application. For example, the isolated polynucleotide molecule can be in the form of a plasmid capable of expressing the encoded virus in a suitable in vitro host cell, as described in more detail below.

The North American PRRSV N protein sequence is highly conserved, with reported sequences of about 93-100
% Identity with each other. North American and European PRRSV N proteins are about 57-59
% Are identical and share a common structural motif. In general, when comparing PRRS coding sequences and isolates that may be numbered differently for a particular nucleotide or encoded amino acid, identification of the appropriate region is a conserved feature in the subject PRRS strain. This is accomplished quickly by identifying the specific amino acid and aligning it with the reference strain.

Recombinant DNA technology includes a very diverse and powerful molecular biology approach aimed at modifying nucleic acids at the DNA level, allowing the genome to be analyzed and modified at the molecular level. In this regard, viruses such as PRRS virus are particularly amenable to the above operations because of their moderate genome size. However, these viruses do not replicate in D
Recombinant DNA technology is not immediately applicable to non-retroviral RNA viruses because it does not contain NA intermediate steps. For such viruses, infectious cDNA clones must be developed before recombinant DNA technology can be applied to their genomes to produce modified viruses. The infectious clone is the full length (genomic length) cD of the virus under test
Can be derived from the structure of NA (here used in the broad sense of a DNA copy of RNA, and not only in the strict sense of a DNA copy of mRNA), and then in vivo where the infectious transcript was introduced into the full length cDNA Synthesized in cells, but infectious transcription can also be obtained by in vitro transcription from full length cDNA in a plasmid with a prokaryotic promoter with the presence of a transcription mixture, or a ligated partial length containing all viral genomes in vitro It can also be obtained by using a cDNA fragment. In all cases, the transcribed RNA carries all modifications introduced into the cDNA and can be used for further passage of viruses thus modified.

Preparation of infectious clones of European PRRS virus isolates or Lelystad virus is described in US Pat. No. 6,268,199, which is fully incorporated herein by reference. The preparation of infectious cDNA clones of North American PRRS virus isolates designated P129 (Lee et al., 2005; Yoo et al., 2004) is described in US Pat. No. 6,500,662, by reference. All of which are incorporated herein. The sequence of P129 cDNA is disclosed in Genbank accession number AF494042 and US Pat. No. 6,500,662. The following test utilizes such an infectious clone expressed by the CMV immediate early promoter in the context of a plasmid and designated pCMV-S-P129, and is also disclosed in US Pat. No. 6,500,662. . There are other plasmids and promoters suitable for use in the present invention, as described in US Pat. No. 6,500,662.

Given the complete sequence of any translation region of interest and the location of the subject amino acid residues, one skilled in the art need only look at the codon table to devise changes in the specific location desired.

Codons are triplet sequences of nucleotides in mRNA and their corresponding c
Constructs a DNA molecule. A codon is characterized by a uracil base (U) when present in an mRNA molecule, but is characterized by a thymidine base (T) when present in DNA. A simple change to the same amino acid residue in a polynucleotide at a codon does not change the sequence or structure of the encoded polypeptide. It is clear that when a particular trinucleotide sequence states that "encodes" any particular amino acid, one of ordinary skill in the art can recognize that the above table provides a method for identifying the particular nucleotide of interest. It is. By way of example, when a particular three nucleotide sequence encodes lysine, the above table discloses that two possible triplet sequences are AAA and AAG. Glycine is encoded by GGA, GGC, GGT (GGU in the case of RNA) and GGG. In order to change lysine to glycine residue in the encoded protein, AAA or AAG triplets in the encoded nucleic acid can be converted to GGA and GGC.
, GGT or GGG.

As mentioned above, the present invention is directed to the provision of PRRS vaccine strains, and host responses to viruses mediated by the interferon pathway are not down-regulated in the presence of other responses. As described in detail below, there are a variety of modifications to the viral genome that are effective in this regard, particularly those found in ORF1a and combinations thereof as disclosed herein. It should be noted that similar modification points can be found in additional translation regions of the PRRS genome as disclosed herein (see Table 9).

Notably, some other past approach to the modification of PRRS polynucleotides is PR.
It has been successful in attenuating RS viruses, and the exact cause of the resulting attenuation is generally unknown, but can provide suitability for vaccine use. For example, attenuation of virulent PRRS virus by mutating or deleting the NLS-2 region, NoLS region, or NES region in the viral nucleocapsid or N protein (encoded by ORF7), translation region 7 (ORF7) It is disclosed to include a deletion in In another aspect, the deletion of ORF7 is within the sequence encoding the nuclear localization signal (NLS) of the capsid protein. OR within an array encoding NLS
The deletion of F7 can include a deletion of one or more amino acids at positions 43-48, or position 4
Amino acid deletions at one or both of 3 and 44 may be included. See, for example, the entire disclosure of US Pat. No. 7,544,362, incorporated by reference. The PRRSV nucleocapsid protein encoded by ORF7 is a small basic protein that is phosphorylated (Woughton, Rowland, and Yoo, 2002) and forms a homodimer (Woughton and Yoo, 2003). A crystal structure has recently been identified (Doan and Dokland, 2003). The N protein appears to have multiple functions in infected cells. In addition to the process that occurs in the cytoplasm, forming a spherical capsid structure with genomic RNA packaged inside, part of the N protein is transported to the nucleus and specifically to the nucleus of the infected cell. The amino acid sequence of the N protein consists of two nuclear localization signals (NLS), a nucleolar localization signal (NoLS), and a nuclear export signal (NE
S) to facilitate transport to and export from the nucleus and nucleolus, respectively (Ro
wland et al. , 1999; Rowland et al. , 2003; Ro
wland and Yoo, 2003). While in the nucleolus, the N protein interacts with the small nucleolar RNA-related protein fibralin and can control rRNA processing and ribosome biosynthesis in infected cells to help virus replication (Yoo et al., 2003).
). This type of viral mutation is valuable in devising a new PRRS vaccine, either alone or in combination with other attenuated mutations. In another example of attenuated PRRS virus, a modification to ORF1a was used. A deletion of the DNA sequence encoding the antigenic epitope between amino acids 616 and 752 in the nonstructural protein 2 hypervariable region encoding the region of ORF1a was used. See U.S. Pat. No. 7,618,797, which is fully incorporated by reference.

Studies on the immunobiology of PRRS virus suggest that the interaction between PRRS virus and PDC deserves testing. This cell type represents 0.2% to 0.8% of human, mouse, rat, pig and monkey peripheral blood mononuclear cells. Despite being rare, this cell is an important component of the innate immune system and is capable of secreting large amounts of IFN-α following viral stimulation. PDCs play a major role in controlling antiviral natural and adaptive immunity because they promote the function of natural killer cells, B cells, and T cells, via secretion of IFN-α. . Furthermore, porcine monocyte-induced dendritic cell maturation (M
oDC) is facilitated by IFN-α secreted by PDC, resulting in an improved ability of MoDC to present antigen and activated T cells. At a later stage of viral infection, PDC differentiates into a unique type of mature dendritic cell that directly regulates T cell function and regulates T cell differentiation.
Directed to cells capable of secreting IFN-γ, a major mediator of antiviral immunity against viruses, including viruses. Of course, there are human viruses, such as respiratory rash virus and measles virus, which are known to suppress the ability of PDC to secrete IFN-α. This suppressive effect plays a role in the humoral immune response and the associated immunopathological dominance observed as a result of infection with these viruses and in the host's susceptibility to second bacterial and viral infections. It seems to fulfill.

In contrast, the new P129-PK-FL and P129-PK-d43 / 44 virus stocks (see below) showed minimal to no suppressive effects on this PDC function, while wild-type PRRSV isolates also showed Ingelvac Similar to both PRRS vaccine strains (see Example 5 and below), purified porcine PDC populations suppressed the ability to produce IFN-α.
The importance of these observations is due in part to the importance of IFN-α in suppressing the development of an adaptive immune response to the virus. Therefore, attenuated viral vaccines derived from viruses that minimize IFN-α are likely to elicit a strong antiviral protective immune response. Adjuvant effect of IFN-α on Ingelvac PRRS MLV vaccine induces virus-specific T cell-mediated IFN-γ response, and the intensity of virus-specific T cell-mediated IFN-γ response elicited by the vaccine is subject to field and laboratory conditions Previously demonstrated to have a positive correlation with protective immunity against the underlying virus. Therefore,
Without being limited by theory, the level of cell-mediated immune response and non-IFN-α-suppressed PRRSV-induced protective immunity is significantly higher than that of the PRRSV isolate exhibiting the wild-type (IFN-α-suppressed) phenotype It is reasonable to expect.

Referring to the present invention, it should be noted that the P129-PK-FL virus is pCMV-S-
Like all five deletion mutants derived from the P129-PK infectious cDNA clone, it lost the ability to suppress IFN-α production. Thus, this abnormal phenotype is not the result of deletion alone, but at least in part, the result of genetic changes fixed during the construction of infectious clones. Interestingly, the uncloned P129 virus serially passaged 63 times on PK-9 cells maintained the ability to suppress IFN induction (Table 1). The most promising explanation for the common IFN phenotype found in all infectious clone-derived viruses is the incorporation of one or more mutations during the generation of infectious clones. These mutations are within the viral RNA used to construct the infectious clone,
Maybe existed at a lower level. Ultimately, the mutation could have been present in the original (passage 0) virus in pigs, or was generated and concentrated during the process of adapting the virus to growth over 16 passages on PK-9 cells. Obtained. The possibility that the mutation is a consequence PCR-induced error or clonal artifact cannot be ruled out. At least the mutation is responsible for the lack of IFN-α inhibitory function being “fixed” during infectious clone construction and must be present in all viruses derived from this particular infectious clone. It is done.

The possibility that the mutation responsible for the altered IFN-α inhibitory phenotype was previously present in the viral RNA used for cDNA clone construction is that PRRSV is a viral RNA-dependent RNA
Considering what is known to easily generate random genetic diversity as a result of errors by polymerase, it is plausible. Virus pseudospecies are composed of heterogeneous mixtures of closely related gene variants that appear naturally during viral replication in vivo. More relevant is the observation of virus pseudospecies after multiple passages of PRRSV derived from infectious cDNA clones in vitro. This is noteworthy because the starting population of the viral genome in previous studies consisted of genetically homogeneous populations, and sequence diversity was rapidly generated during passage in cell culture. In the current study, the original P129 virus has not been cloned (biologically or molecularly) before passage 16PK-9, leading to the construction of infectious clones, so the level of genomic heterogeneity is Can be higher. Therefore, among the pseudo-species responsible for the loss of the IFN-α inhibitory function, the selection of the possibility of incorporation into the PRRSV RNA mutant and the pCMV-S-P129-PK17-FL infectious cDNA clone is plausible. The incorporation of these mutations into infectious clones shares this unique biological phenotype that all derived viruses can prove to be important for the development of effective next generation PRRS vaccines. Can be accidental under some conditions.

Wild-type PRRS virus strain P129, like other strains of this virus, strongly suppresses the ability of peripheral blood mononuclear cells (PBMC) and plasmacytoid dendritic cells (PDC) to produce interferon (IFN) -α. Showed the effect. On the other hand, P129 (pCMV-S-P129
Virus derived from the infectious cDNA clone of (-PK17-FL) showed a significant attenuation in the IFN-α-inhibited phenotype. This infectious clone was constructed from a virus previously adapted for culture over 16 consecutive passages in the CD163 expressing porcine kidney cell line PK-9 (US Pat. No. 7,754, fully incorporated by reference). 464). P129-PK-FL and P129-PK-d43 / 44 IFN-α inhibitory phenotypes range from low to negligible, both of which have high inhibitory properties.
In marked contrast to that exhibited by any of the gelvac PRRS modified live virus vaccine strains. These results indicate that P129-PK-FL and P129-PK-d4
3/44 viruses are biologically parental low passage P129 isolates, other wild type PRRS viruses,
And the Ingelvac PRRS vaccine are different. Attenuation I
The potential implications of the FN-α inhibitory phenotype are described as well as possible reasons for phenotypic changes.
Amino acid modification of the present invention

In accordance with the practice of the present invention, a new isolate of PRRS contains an amino acid at a specific position in the protein encoded from ORF1, in either North American or Chinese genotypes, which allows the desired phenotype to be expressed in these viruses. Can be identified in the field given. In another example, as described above,
Modify the gene sequence (and thus the amino acid sequence) of the encoded ORF1 protein, again producing modified North American and Chinese PRRS viruses, and infectious clones, and vaccines thereof, all of which have such a phenotype Standard genetic procedures can be used for this purpose. In a preferred embodiment, the phenotype induces an attenuated interferon-alpha inhibitory effect compared to wild-type PRRS virus and, optionally, a host disease that is almost undetectable but a strong immune response. Including but not limited to the ability to breed or persist in animals (pigs).

As a result, in the practice of the present invention, North American PRRS strains or isolates that may serve as a starting point are disclosed in, for example, U.S. Pat. Nos. 6,500,662, 7,618,797,
7,691,389, 7,132,106, 6,773,908, 7,264,957, 5,695,766, 5,476 778, No. 5,846,805, No. 6,042,830, No. 6,982,160, No. 6
241, 990, and 6,110,468. See, for example, published Chinese application CN201633909 for TJM-92 virus to Chinese application CN200910091233.6 for Chinese PRRS strains and isolates that may serve as a starting point.

For the following discussion, the internationally recognized single and three letter names are the most common amino acids encoded by DNA, alanine, (Ala, A), arginine (A
rg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (
Gly, G), histidine, (His, H), isoleucine (Ile, I), leucine (
Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (
Phe, F), proline (Pro, P), serine (ser, S), threonine (Thr,
T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val,
V).

Tables 9 and 10 identify the observed amino acid changes responsible for attenuated toxicity in North American (and Chinese) PRRS that correlate with suppressed attenuation of interferon alpha activity. The result is a safe and strong immune response to the vaccine. Table 10 identifies very favorable amino acid corrections in this respect within ORF1a and shows how these mutations occurred with respect to passage from other P129 cultures (and this history test is It should be noted that, if necessary, it facilitates the devising of mutagenesis strategies to (re-) construct the encoded DNA having any of the amino acid changes of the present invention. In this regard, OR
The most favorable amino acid improvement to F1a (as demonstrated at P129 passage 52, 18
2 asparagine, 189 asparagine, 273 tyrosine, 302 histidine, 6
65 threonine, 943 cysteine, 1429 threonine, 1505 alanine,
It contains 2410 asparagine and can potentially modify protein function further, adding potentially many glycosylation opportunities.

Accordingly, the present invention provides an isolated porcine reproductive and respiratory syndrome virus (PRRS), wherein the protein encoded by ORF1a comprises an amino acid sequence comprising any of the following:

Amino acid N in amino acid sequence AMA N VYD (SEQ ID NO: 9),
Amino acid sequence IGH N AVM in amino acid N (SEQ ID NO: 12),
Amino acid D in the amino acid sequence TVP D GNC (SEQ ID NO: 15),
Amino acid Y (SEQ ID NO: 18) in amino acid sequence CWW Y LFD;
Amino acid H (SEQ ID NO: 21) in the amino acid sequence HGV H GKY,
Amino acid V (SEQ ID NO: 24) in the amino acid sequence AAK V DQY,
Amino acid T in the amino acid sequence PSA T DTS (SEQ ID NO: 27)
Amino acid L in the amino acid sequence LNS L LSK (SEQ ID NO: 30)
Amino acid C in the amino acid sequence APM C QDE (SEQ ID NO: 33),
Amino acid T (SEQ ID NO: 36) in the amino acid sequence CAP T GMD;
Amino acid A in the amino acid sequence PKV A KVS (SEQ ID NO: 39),
Amino acid I (SEQ ID NO: 42) in the amino acid sequence AGE I VGV;
Amino acid N (SEQ ID NO: 45) in the amino acid sequence ADF N PEK and amino acid I (SEQ ID NO: 48) in the amino acid sequence QTP I LGR.

More specifically, the present invention provides isolated porcine reproductive and respiratory syndrome virus (PRRS), wherein the protein encoded by ORF1a comprises an amino acid sequence comprising any of the following: Is done.
Amino acid N in amino acid sequence A N V (see SEQ ID NO: 9),
(See SEQ ID NO: 12) amino acid N in the amino acid sequence H N A,
(See SEQ ID NO: 15) amino acid D in the amino acid sequence P D G,
Amino acid Y in amino acid sequence W Y L (see SEQ ID NO: 18);
Amino acid H in the amino acid sequence V H G (see SEQ ID NO: 21),
Amino acid V in the amino acid sequence K V D (see SEQ ID NO: 24),
Amino acid T in amino acid sequence A TD (see SEQ ID NO: 27);
Amino acid L in amino acid sequence S L L (see SEQ ID NO: 30);
Amino acid C in amino acid sequence M C Q (see SEQ ID NO: 33);
(See SEQ ID NO: 36) amino acid T in the amino acid sequence P T G,
Amino acid A in amino acid sequence V A K (see SEQ ID NO: 39),
(See SEQ ID NO: 42) amino acid I in the amino acid sequence E I V,
Amino acid N in the amino acid sequence F N P (see SEQ ID NO: 45) and Amino acid I in the amino acid sequence P I L (see SEQ ID NO: 48).

  As mentioned above, there are well-known strains and isolates of North American or Chinese PRRS. And new strains will evolve or continue to be separated. Although there is a high level of amino acid sequence homology between all these strains, those skilled in the art will immediately recognize that there are some variants, and certainly take advantage of these differences and similarities, The phenotypic characteristics of the vaccine strain can be further improved.

First, for all of the amino acid motifs defined by SEQ ID NO as specified directly above (pages 27-28) by SEQ ID NO: underlined preferred amino acids (as provided from P129 passage 52) Generally remains completely useful even if adjacent amino acids are otherwise altered from the specified SEQ ID NO sequence. Thus, with respect to AMA N VYD (SEQ ID NO: 9), as a specific and representative example, generally examine the protein sequence expressed by the corresponding ORF1 from any North American or Chinese PRRS to find the corresponding amino acid motif It is possible even if additional changes have occurred in such other strains as a result of substitutions and / or developments that cause deletions or additions. As will be appreciated by those skilled in the art, the preferred amino acid change demonstrated at P129 passage 52 is therefore also in the overall amino acid sequence that is directly 5 'or 3' to the designated amino acid of passage 52. It remains operable despite other changes. This is especially true when the comparative amino acid changes are considered conservative. Thus, for AMA N VYD (SEQ ID NO: 9) and its subsequence ANV, for example, when valine therein is replaced with isoleucine, or leucine, or any other residue, or the residue is simply missing. If additional or additional residues are added, it should be readily possible to identify comparable motifs in other PRRS strains. There are a number of computer programs to identify alignments and thus determine whether a polypeptide sequence motif corresponds to, for example, the so-called Blosum table (based on a certain level of percent identity). S. Henikoff et al. “Amino Acid Substitution metrics from protein blocks”, Proc Natl Acad Sci, USA, 89 (22), pp. See 10915-10919, Nov 15, 1992. A. L. Lehninger et al. See also Principles of Biochemistry, 2005, MacMillan and Company, 4th edition. In addition, conservative amino acid changes are classified into five general groups: sulfhydryl group (Cys), aromatic (Phe, Tyr, and Trp), basic (Lys, Arg, His), and fatty (Val , Ileu, Leu, Met) and hydrophilicity (Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser and Thr). Thus, any one or more other amino acids adjacent to the designated position may be added, deleted or substituted, and any of the amino acid changes specified in P129 passage 52 will be performed at the appropriate and corresponding positions. It is within the practice of the present invention to modify any North American or Chinese PRRS encoding nucleotide sequence for incorporation.

Further, based on similar principles, one of ordinary skill in the art, once identified a suitable amino acid from a particular passage 52 change identified for ORF1a by practice of the present invention, will be able to identify any such passage 52 amino acid. It will be appreciated that a conservative exchange can be used with the P129 mutant, or any other North American or Chinese strain, with substantial preservation of the intended passage 52 phenotype. Thus, as a representative example, for S L L (within SEQ ID NO: 30), the designated leucine residue can be further substituted with isoleucine, valine or methionine, and with respect to F N P (within SEQ ID NO: 45). The designated asparagine can be replaced with any of Ala, Pro, Gly, Glu, Asp, Gln, Ser and Thr, and for V A K (see SEQ ID NO: 39), the designated alanine is Asn, Pro, Gly, Glu, Asp, Gln, Ser and Thr can be substituted, all and similar, but unique passaged 52 amino acids from which any such substituted amino acids were originally identified It will be readily appreciated that changing and functioning similarly at a specific location is not required by the present invention. Of course, the use of standard conservative amino acid changes by any other recognized model is implemented in the present invention. For example, including the reverse of all cases, Asp for Glu and vice versa, Asn for Gln, Arg for Lys, Ser for Cys or Thr, Phe, Leu or Ileu for Tyr Val, Ala for Gly, and the like.

  Furthermore, within the practice of the invention, placing any of the individual passage 52 amino acid changes (as identified for ORF1a above) in any North American China PRRS with the desired phenotypic effect. However, it is more preferred to include as many amino acid choices as Table 9 or Table 10 as possible, as provided in the final construct, usually by appropriate modification of the encoding polynucleotide sequence. Thus, practice of the present invention provides a Chinese or North American PRRS virus (and corresponding) that provides any of the identified passage 52 ORF1a alterations up to 2, 3, 4, 5, 6, 7, 8, 9, and up to about 17. (Table 9) Every specific all of the comprehensively identified passage 52 amino acid changes, three sets or another higher in the final viral sequence Includes combinations. Such amino acid changes can, of course, introduce the corresponding coding nucleotide sequences of the virus by site-directed mutagenesis, PCR, and other techniques well known in the art.

To demonstrate that a particular genetically engineered strain is attenuated, the experiments described below can be used.

Each study includes at least 10 heifers per group obtained from a farm without PRRSV. Animals lack PRRS virus specific serum antibodies and PRRS
Tested for V-negative. All animals included in the study are of the same source and type.
The distribution of animals to the group is randomly selected.

Antigen administration is performed on day 90 of pregnancy by intranasal administration of 1 ml PRRSV at 10 ⁵ TCID ₅₀ per nostril. There are at least three groups for each test configuration, one group for P129 challenge, one group for test challenge with potentially attenuated virus, and one group for strict control. is there.

As the strict control group continues to show PRRSV negative over the time course of the test, the test is considered effective and live healthy piglets born in the group challenged with P129 are strict At least 25% less than the normal control group.

Attenuation, in other words low toxicity, is defined as a statistically significant change in one or more parameters that determine reproductive behavior or other symptomatology.
A significant decrease in at least one of the following parameters of the test group (a virus that may be attenuated) compared to the unmodified parent strain infection group is an indication of attenuation.
a) Frequency of stillbirth b) Miscarriage at 112 days of gestation or earlier c) Number of mummy piglets d) Number of weak and weak piglets e) Preweaning mortality and comparison with unmodified parental infection group A significant increase in at least one of the following parameters of the tested group is preferred.
f) Number of piglets weaned per sow g) In a number of live healthy piglets born per sow, respiratory symptoms and other symptoms of PRRSV infection are attenuated Could be examined to establish.

Attenuated strains are valuable for vaccine formulation. Current vaccines are effective if they protect pigs from infection with PRRS virus. After administration of the vaccine to one or more unaffected pigs, a biologically pure virus isolate (eg VR2385, VR2386
, P129, etc.), in general, in any unaided similar pig, compared to those changes or symptoms caused by the isolate (ie in relation to appropriate controls) Vaccines protect pigs from infection with PRRS virus if they result in a reduction in the severity of symptoms or histopathological changes (eg lung lesions) and / or disease symptoms. More specifically, current vaccines administer the vaccine to one or more suitable pigs as needed, and then after a suitable length of time (eg, 4 weeks), biologically pure PRRS
It can be shown to be effective by challenge with a large sample of V isolates (10 ^(3-7) TCID ₍₅₀₎ ). The blood sample is then withdrawn from the challenged pig after approximately one week, and then an attempt is made to separate the virus from the blood sample. Separation of attenuated amounts of virus (or no virus) is an indication that the vaccine can be effective, while isolation of large amounts of virus is an indication that the vaccine cannot be effective.

Thus, the effectiveness of current vaccines can be quantitative (ie, a reduction in the percentage of cirrhotic lung tissue compared to the appropriate control group) or qualitatively (eg, PRRSV from blood
, Detection of PRRSV antigen by immunoassay in lung, tonsil or lymph node tissue samples. Symptoms of porcine reproductive and respiratory disease can be quantitative (eg body temperature / fever) or semi-quantitative (eg presence or absence of one or more symptoms or one or more symptoms such as cyanosis, pneumonia, lung disorders etc. (Attenuation of severity).

Unaffected pigs are those who have not been exposed to porcine reproductive and respiratory disease and respiratory disease infectious agents, or who have been exposed to porcine reproductive and respiratory disease and respiratory disease infectious agents, but the symptoms of the disease It is a pig that does not show. Affected pigs exhibit symptoms of PRRS or are able to isolate PRRS.

The vaccines of the present invention can be formulated according to the following acceptable practices to include an acceptable carrier for animals, including humans (if applicable). For example, standard buffers, stabilizers, diluents, preservatives, and / or solubilizers. It can also be formulated to promote sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, glucose, mannitol, sorbitol and lactose, among others.
Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly beneficial when formulating modified live vaccines, are well known or apparent to those skilled in the art. Remington's P, incorporated herein by reference.
hermetic science, 18th ed. , 1990, Mack
See Publishing.

The vaccine of the present invention further comprises, for example, an adjuvant or cytokine, among others,
One or more additional immunomodulatory components can be included. Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include, for example, the RIBI adjuvant system (Ribi Inc., Hamilton)
n, Mont. ), Alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta, Ga.), QS- 21 (Cambridge B
iotech Inc. , Cambridge Mass. ), SAF-M (Chiro
n, Emeryville Calif. ), AMPHIGEN. RTM adjuvant,
Includes saponin, Quil A or other saponin fragments, monophosphoryl lipid A, and an abridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful with the vaccines of the present invention include modified SEAM62 and SEAM1 / 2 formulations. Modified SEAM62 is 5% (v / v) squalene (Sigma), 1% (v / v) SPA.
N. RTM85. Detergent (ICI surfactant), 0.7% (v / v) TWEEN. RTM
. 80 detergent (ICI surfactant), 2.5% (v / v) ethanol, 200 pg / ml
Quil A, 100 [mgr] g / ml cholesterol, and 0.5% (v / v)
It is an oil-in-water emulsion containing the lecithin. Modified SEAM1 / 2 is 5% (v / v) squalene, 1% (v / v) SPAN. RTM85. Detergent (ICI surfactant), 0.
7% (v / v) TWEEN 80 detergent, 2.5% (v / v) ethanol, 100. mu
. g / ml Quil A, 50. mu. An oil-in-water emulsion containing g / ml cholesterol. Other immunomodulators can be included in vaccines containing, for example, one or more interleukins, interferons, or other well-known cytokines.

The vaccine of the invention can optionally be formulated for sustained release of viruses, infectious RNA molecules, plasmids, or viral vectors of the invention. Examples of such sustained release formulations are, for example, viruses, infectious RNA molecules, plasmids or biocompatible polymers such as polylactic acid, lactic acid-glycolic acid copolymers, methylcellulose, hyaluronic acid, collagen, and the like. Contains viral vectors combined with the body. The structure, selection, and use of degradable polymers in drug delivery vehicles are incorporated herein by reference. Domb et. al. , Polymers for Advanced T
has been reviewed in several publications, including technologies 3: 279-292. Additional guidance on selecting and using polymers in pharmaceutical formulations can be found in texts known in the art. For example, M.M. Chasin and R.M. Langer
(Eds), 1990, Drugs and the Pharmaceutical.
Sciences, Vol. 45, M.M. Dekker, N .; Y's “Biodegrad”
“able Polymers as Drug Delivery Systems”, which is incorporated herein by reference. Alternatively, or in addition, viruses, plasmids, or viral vectors can be microencapsulated to improve administration and efficacy. Are well known in the art, for example, U.S. Pat. No. 3,137,631, U.S. Pat. No. 3,959,457, U.S. Pat. No. 4,205,060, U.S. Pat. Including the techniques described in US Pat. No. 4,606,940, US Pat. No. 4,744,933, US Pat. No. 5,132,117 and International Patent Publication No. WO 95/28227, all of which are referenced Is incorporated herein by reference.

Liposomes can also be used to deliver sustained release of viruses, plasmids, or viral vectors. Details regarding how to make and use liposomal formulations are described in particular in US Pat. No. 4,016,100, US Pat. No. 4,452,747, US Pat.
921,706, US Pat. No. 4,927,637, US Pat. No. 4,944,948, US Pat. No. 5,008,050, and US Pat. No. 5,009,956. All of which are incorporated herein by reference.

  An effective amount of any of the above vaccines can be determined by conventional methods. Starting with a low dose of virus, viral protein plasmid or viral vector, the dose can then be increased while monitoring the effect. An effective amount can be obtained after a single dose of vaccine, or after multiple doses of vaccine. Well known factors can be taken into account when determining one optimal dose per animal. These include animal type, size, age and general condition, the presence of other drugs in the animal and the like. The actual dosage is preferably selected after considering the results of other animal experiments. (See, eg, Example 2 and Example 3 below).

  One way to detect whether an adequate immune response has been achieved is to determine seroconversion and antibody titers in the animals after vaccination. The timing of vaccination and the number of boosts, if any, are preferably determined by a physician or veterinarian based on an analysis of all relevant factors, some of which are described above.

Effective doses of the viruses, proteins, infectious DNA molecules, plasmids or viral vectors of the invention are determined using well-known techniques, taking into account factors that can be determined by those skilled in the art, such as the weight of the animal to be vaccinated. Can be determined. The dosage of the virus of the present invention in the vaccine of the present invention is preferably about 10 ¹ to about 10 ⁹ pfu (plaque forming units), more preferably about 10 ² to about 10 ⁸ pfu, most preferably about It is in the range of 10 ³ to about 10 ⁷ pfu. The dosage of the plasmid of the present invention in the vaccine of the present invention is preferably in the range of about 0.1 mg to about 100 mg, more preferably about 1 to about 10 mg, and even more preferably about 10 to about 1 mg. The dosage of the infectious DNA molecule of the present invention in the vaccine of the present invention is preferably in the range of about 0.1 mg to about 100 mg, more preferably about 1 to about 10 mg, more preferably about 10 to about 1 mg. It is. The dosage of the viral vector of the present invention in the vaccine of the present invention is preferably about 10 ¹ to about 10 ⁹ pfu, more preferably about 10 ² to about 10 ⁸ pfu, more preferably about 10 ³ to about 10 The range is 10 ⁷ pfu. Suitable dosage sizes range from about 0.5 ml to about 10 ml, more preferably from about 1 ml to about 5 ml.

  Suitable dosages for viral protein or peptide vaccines in accordance with the practice of the present invention are generally in the range of 1-50 micrograms per dose, or an amount of adjuvant to be determined in a recognized manner for each such substance. At higher amounts than can be determined by standard methods. In a preferred embodiment of the present invention relating to pig vaccination, the optimal age target for animals is about 1-21 days. Also, during that day, other scheduled vaccinations such as Mycoplasma hyopneumoniae or PCV can be accommodated before weaning. In addition, a suitable schedule of vaccination for breeding sows includes similar doses with an annual revaccination schedule.

An effective amount of any of the above vaccines is determined by conventional methods. Starting with a low dose of virus, plasmid or viral vector, the dose can then be increased while monitoring the effect. An effective amount can be obtained after a single dose of vaccine, or after multiple doses of vaccine. Well known factors can be taken into account when determining one optimal dose per animal. These include animal type, size, age and general condition, the presence of other drugs in the animal and the like. The actual dosage is preferably selected after considering the results of other animal experiments.

One way to detect whether an adequate immune response has been achieved is to determine seroconversion and antibody titers in the animals after vaccination. The timing of vaccination and the number of boosts, if any, are preferably determined by a physician or veterinarian based on an analysis of all relevant factors, as some of the above show.

An effective amount of a virus, infectious RNA molecule, plasmid or viral vector of the invention can be determined using well-known techniques, taking into account factors that can be determined by one skilled in the art, such as the weight of the animal to be vaccinated. . By way of example, the vaccine may be delivered orally, parenterally, intradermally, subcutaneously, intramuscularly, intranasally or intravenously. Oral delivery can include, for example, adding the composition to an animal feed or drink. Factors related to vaccine dosage include, for example, pig weight and age.

The present invention further includes PRRS viruses, infectious RNA molecules, plasmids, as described herein,
Alternatively, synthesis of an effective amount of one of the PRRS viruses, infectious RNA molecules, plasmids, or viral vectors of the present invention in a method acceptable for preparing a vaccine comprising a viral vector, pharmaceutical or veterinary use. A method comprising:

In addition, the live attenuated vaccines of the present invention can be used to encode heterologous antigenic epitopes that are inserted into the PRRS virus genome using well-known recombinant techniques, as described in US Pat. No. 6,500,662. Can be modified. See also U.S. Pat. No. 7,132,106, which is fully incorporated by reference. Antigenic epitopes useful as heterologous antigenic epitopes for the present invention are porcine parvovirus, porcine circovirus, porcine rotavirus, swine influenza, pseudorabies virus, infectious gastroenteritis virus, porcine respiratory coronavirus, porcine cholera Virus, African swine fever virus, Encephalomyocarditis virus, Porcine paramyxovirus, Torque tenovirus, Actinobacillus pleurone pneumonia, Actinobacillus swiss, Bacillus anthracis, bronchial sepsis, Clostridium hemolyticum, Clostridium perfringens, tetanus , Escherichia coli, swine erysipelas, parainfluenza, leptospira, mycoplasma hyopneumoniae, mycoplasma hyorinis, mycoplasma hyosinobie, pasturela maltosida, swine fever, murine Skins,
It includes antigenic epitopes from porcine pathogens other than PRRS virus, including but not limited to antigenic epitopes from porcine pathogens selected from the group consisting of Streptococcus equis myris and Streptococcus pig. Nucleotide sequences encoding antigenic epitopes from the aforementioned porcine pathogens are well known in the art and are available from public genetic databases, such as Genbank from the National Center for Biotechnology Information on the World Wide Web (USA). You can get it.

Additional features and variations of the present invention will become apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are contemplated as aspects of the present invention. Similarly, inventive features described herein may also include additional embodiments contemplated as aspects of the invention, regardless of whether a combination of features is explicitly referred to above as aspects or embodiments of the invention. Can be recombined. Also, only such limitations as described herein that are important to the invention should be viewed as such. Variations of the invention that are lacking in limitations not described herein to be important are contemplated as aspects of the invention. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

Many modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.

  The following examples are intended to illustrate but not limit the present invention.

Example 1
PRRSV adaptation and attenuation separates P129 into PK-9 cells.

The toxic PRRS isolate P129 was founded in 1995 in Animal Disease Dia.
Isolated from sick pigs in Indiana at gnostic Laboratory of Purdue University. Serum samples from this pig were passaged once in a high health pig that swells the serum and lung homogenate stock. Viral RNA was extracted from serum and lung homogenates and used to determine the described complete genomic consensus sequence for P129 passage 0 virus. RNA was first stimulated with random hexamers and used to integrate the cDNA. Genome is high fidelity (proofreading) PC
Using R, it was amplified into 3 overlapping fragments. 3 separate PCR reactions (1
The PCR product from (per genome segment) was a T / A clone and was used to generate a full-length genomic consensus sequence for the sequence of DNA (see SEQ ID NO: 1).

An aliquot of the same porcine serum used for the DNA sequence contained P129 passage 0 and was used initially in infecting porcine alveolar macrophage (PAM) cells. P
Progeny virus from AM infection (passage 1) was filtered through a 0.1 micrometer dropper filter and used to infect PK-9 cells.

PK-9 cells are a transgenic cell line derived by stably transfecting the PK0809 porcine kidney cell line with a plasmid encoding the deleted version of the porcine CD163 gene and the neomycin resistance gene. Construction details and PK
The specialization of the -9 cell line has been described previously.

Adaptation of passage 1 virus from PAM cells to growth on PK-9 cells was difficult and required several attempts to have multiple parallel lines. Infection was detected by viral nucleocapsid protein (Rural Technology) by immunofluorescence of replication wells.
es Inc, Brookings South Dakota), FI
This was monitored using the TC-conjugated monoclonal antibody SDOW17. Early passage resulted in several small growth foci but did not produce enough acellular virions to be able to cause new monolayer infections. These passages are called Accutase
By treating the infected monolayer with (trypsin substitute) and repopulating cells in multiple wells with fresh medium, with or without the addition of uninfected PK-9 cells
Realized. After several such passages, some lines showed a clear increase in the frequency and size of the fluorescent growth foci. Some of these acquired the ability to be passaged using a cell-free virus fluid. Passage 17 (1 on PAM cells, 16 on PK-9) ensures that one lineage is maintained using dilutions of cell-free fluid from previous passages, and within a few days the entire unit is maintained. Infected the molecular layer. The virus did not cause a cytopathic effect on PK-9 cells at any passage level. RNA was extracted from infected PK-9 cells at virus passage 17 and used to construct infectious cDNA clones.

Example 2
Construction of infectious cDNA clone of P129-PK passage 17

An infectious cDNA clone of P129-PK passage 17 virus was constructed using a backbone plasmid as described above. The viral genome is reverse transcribed and PCR in three overlapping segments, with unique restriction endonuclease sites in the overlapping region occurring naturally.
Amplified by. Products from three separate PCR reactions were cloned, sequenced and aligned to generate consensus sequences for each genomic segment. If none of the three cloned products of a given segment match the predicted consensus amino acid sequence for that segment, one of the clones until the predicted consensus amino acid sequence matches. Was altered by subcloning and / or site-directed mutagenesis. The three genomic segments and the plasmid backbone were joined using standard cloning techniques and restriction endonuclease sites. When nucleic acid was introduced into PK-9 cells, the resulting full-length clone designated pCMV-S-P129-PK17-FL became infected. The sequence of this infectious cDNA clone is given in SEQ ID NO: 2. The genome is essentially the same as passage 17 virus constructed at the standard end, and any insertions or deletions associated with passage 17 consensus sequences are missing. There are no designed restriction sites or other targeted changes in the viral genome of this infectious cDNA clone.

Complete genomic consensus sequence of P129 passage 0 and infectious clone pCMV-SP
The nucleotide and amino acid differences between the genomic sequences of 129-PK17-FL are listed individually in Table 6. Table 6 includes all nucleotide differences and resulting amino acid differences by genomic position. A subset of these mutations range from IFN suppression (passage 0) to IFN
It is responsible for the change in phenotype to non-suppressed (all viruses obtained from passage 17 infectious cDNA clones). Table 7 summarizes the nucleotide, amino acid, and non-conserved amino acid differences due to the PRRSV translation region (ORF) or nonstructural protein (nsp). For the purposes of Table 7, the following groups of amino acids are among those thought to be conserved: [K, R], [D, E], [L, I, V, A] , And [S, T].

Example 3
Deletion mutant in P129-PK passage 17

Deletions in two regions of the genome were designed into the infectious cDNA clone pCMV-S-P129-PK17-FL to generate five transgenic infectious clones.

One region of the genome that causes modification is the nucleocapsid protein (PRRSV OR
Nuclear localization sequence (NLS) located at amino acid positions 41-47 of (encoded by F7)
Met. Two types of deletions were made. These deletions were described above in the context of another PRRSV infectious clone. The wild type sequence of amino acid residues 41-49 is PG. . . KN
It is. Also, in mutant “d43 / 44”, known as “PG... KS”,
Lysine residues 43 and 44 are deleted and asparagine residue 49 is changed to serine. Also,
In the variant “d43 / 44/46”, known as “PG--S-KS”, lysine residues 43, 44 and 46 are deleted and asparagine residue 49 is changed to serine. Infectious clones obtained from pCMV-S-P129-PK17-FL that incorporate these deletions are pCMV-S-P129-PK17-d43 / 44 and pCMV-S, respectively.
-P129-PK17-d43 / 44/46. See U.S. Patent No. 7,544,362.

The second region of the genome that caused the modification was present in the hypervariable region of nsp2 in ORF1a. The deletion of 131 amino acids (393 nucleotides) was described above in the context of another PRRSV infectious clone. PCMV-S-P129-P, which incorporates this deletion
The infectious clone obtained from K17-FL is pCMV-S-P129-PK17-ns
p2.

Infectious clones that combine NLS and nsp2 deletions are also pCMV-S-P129.
Generated in the PK17-FL backbone and these are pCMV-S-P129
-PK17-nsp2-d43 / 44 and pCMV-S-P129-PK17-nsp
2-d43 / 44/46.

Example 4
Virus generation and growth in PK-9 cells

The six infectious clones described in Example 3 were nucleic acid introduced into PK-9 cells to produce 6 viruses, as shown in Table 1. Virus was generated from these infectious clones using Lipofectamine 2000 by direct nucleic acid introduction of a circular plasmid into PK-9 cells. Following nucleic acid transfer, the recovered virus was serially passaged again in PK-9 cells to further increase titer and attenuate toxicity. Stocks were created as vaccine candidates for in vitro testing and in vivo evaluation. Unmodified pCMV-
In the case of P129-PK17-FL virus obtained from the S-P129-PK17-FL infectious clone, the virus was cultured until a total of 52 passages from pigs were reached. The complete genome of this virus was sequenced at passages 24 (SEQ ID NO: 3) and 52 (SEQ ID NO: 6).

Example 5
Virus obtained from P129-PK passage 17 infectious CDNA clone attenuates its ability to suppress IFN-alpha induction

Viruses and cells. MARC-145 and ST cells contain 5% fetal calf serum (FB
S) and antibiotics (50 μg / ml gentamicin, 100 UI penicillin and 1
Grown in modified Eagle's medium (MEM) supplemented with 00 μg / ml streptomycin). Porcine alveolar macrophage ZMAC-1 cells are RPMs supplemented with 10% FBS
I was raised at I-1640. The TGE virus strain Purdue is a 0.
Prepared by infection of confluent ST cell monolayers at a multiplicity of 01. Viral inoculum was removed after 1 hour and cells were cultured in MEM supplemented with 2.5% FBS at 37 ° C. in 5% CO ₂ atmosphere. The virus was released by freezing and thawing the cell monolayer after an 80% cytopathic effect was observed. TGE virus stock was centrifuged at 3,500 rpm at 4 ° C. for 15 minutes and stored at −80 ° C. until use. The virus stock (from PK-9 cell) was as follows. P129-PK-FL and P1
29-PK-dnsp2-d43 / 44/46 was passaged 8/25 (8
25) from pigs. The other four viruses (P129-PK-d43 / 44, P
129-PK-d43 / 44/46, P129-PK-dnsp2, and P129-P
K-dnsp2-d43 / 44) was present at passage 21/38. The working stocks of various PRRS viruses are the commercial vaccines Ingelvac PRRS MLV and I
Prepared by making single passages on ZMAC-1 cells except that ngelvac PRRS ATP was rearranged according to the manufacturer's instructions and used for direct infection.

Isolation of porcine PBMC. Fresh heparinized venous blood was diluted with Hanks buffer and PBMCs were isolated by density centrifugation through a Ficoll Hypac 1077 (Sigma) gradient. After washing with Hanks buffer, cells were interrupted with L-glutamine (Mediatech) supplemented with 5% fetal calf serum (Gibco), 100 U / ml penicillin, 0.
Suspended in RPMI medium with 1 mg / ml streptomycin, 1 mM sodium pyruvate, 1 × non-essential amino acid (Mediatech) 100 U / ml gentamicin and 250 mM 2 mercaptoethanol (Sigma).

Purification of porcine plasmacytoid dendritic cells. Purification of porcine plasmacytoid dendritic cells is as described above (Cal
zada-Nova, submitted) were performed and these cells (Summerfield)
et al. , 2003) based on the characteristic expression of CD4 and CD172. Briefly, fresh porcine PBMC are suspended in PBS with 0.5% BSA and swine C
Labeled with an optimal amount of mAb recognizing D172 (74-22-15, VMRD). 1
Following the second wash, the secondary goat anti-mouse antibody was then injected with PE (Southern Biote
ch), and after washing FITC is anti-CD4 (74-12-4, VMRD)
And cultured in a labeled state. PDCs were classified on a reflective cell sorter (iCyt) and the classification gate was set to the CD4 ⁺ / CD172 ^low population. After sorting, cell purification was confirmed by reanalysis. In all cases, the purity was> 95%.

Test method for cytokine secretion. PBMC or PDC for 16 hours (37 ° C, 5% C
O _2), it was stimulated with different stimulator or false stimulus. After incubation, the medium covering the stimulator cells is a sandwich ELISA prepared with commercially available monoclonal antibodies.
(Anti-pig IFN-α mAb K9 and F17) was measured for the presence of IFN-α. Briefly, Immulon II plates (Dynatech Inc.) were cultured with anti-pig IFN-α mAb F17 (PBL Laboratories) by overnight incubation at 4 ° C. followed by blocking with RPMI medium supplemented with 5% fetal calf serum. Covered. After 1 hour, the media was discarded and 50 microliters of detachment was added to duplicate measurement wells and examined. After 1 hour of incubation, the measurement wells are washed 4 times and then biotinylated, anti-pig IFN-α mAb K9 (PBL Laboratories)
, HRP-conjugated streptavidin (Zymed Laboratories), and TMB substrate (KPL). The optical density was determined with an ELISA plate reader.

Virus derived from the P129-PK passage 17 infectious CDNA clone is IFN-
It lacks the ability to suppress alpha induction. The working virus stock utilizes the porcine alveolar macrophage cell line ZMAC-1 and 4 different PRRS wild type virus isolates (P34).
12, P129, IND5, NADC 20). Additional stock is also available from PK-9, FK. Prepared from the first two wild-type virus derivatives adapted to grow in cell culture by repeated passages in D4 or Mark-145 cells. Three of the four wild type isolates (P129, IND5, NADC20) are ZMAC
-1 grows easily and efficiently in cells, and the P3412 wild type isolate is 10 ⁵ TCID ₅₀
A titer of about 10 ⁷ TCID ₅₀ / ml was reached, compared to a titer of only / ml In particular,
Stocks of P129 virus prepared with the ZMAC-1 cell line reached a titer 10 times higher than that obtained with virus-matched PK-9 or MARC-145 cells.
A test of the ability of these viruses to stimulate IFN-α secretion by PBMC, with one exception (segregating P3412 clone C), with only a small amount of IFN-α (<50 pg).
) Were secreted by these cells in response to their exposure to any of the tested PRRS virus stocks. IFN-α produced by the same cells as a result of their exposure to porcine coronavirus, infectious gastroenteritis virus (TGEv)
Negligible by comparison with the abundant secretion of (17,540 pg).

PRRSV not only makes it impossible to stimulate IFN-α production by porcine PBMC,
Actively suppresses its generation. The suppression effect of PRRSV stock is the presence or absence of PRRSV, T
It was determined by measuring the amount of IFN-α secreted by PBMC in response to exposure to GEv. As shown in Table 2, all four wild-type PRRS virus isolates were examined and PB to TGEv was just as all cell cultures were adapted to the derivatives.
A strong inhibitory effect (> 80%) was shown in the IFN-α reaction of MC. Full length P129-PK-FL
Analysis of a group of virus stocks derived from an infectious cDNA clone (pCMV-S-P129-PK17-FL) containing the virus and several genetically modified deletion mutants was performed. As shown in Table 3, when compared to the strong suppressive effect (95%) of the parental wild type isolate P129 (passage 1), the P129-PK-FL virus, and all deletion mutants, PBMC showed a markedly attenuated ability to suppress IFN- □ induction by TGEv. In order to further evaluate the IFN-α phenotype of these viruses, subsequent experiments were
29-PK-FL and P129-PK-d43 / 44 viruses, parental P129 wild type, and / or Boehringer Ingelheim (Ingelvac PRRS
The focus was on performing a direct comparison between two commercially available modified live PRRSV vaccines generated by MLV and Ingelvac PRRS ATP). An additional low passage reference isolate (NVSL-14) was also examined. As shown in Table 4, in four independent experiments, P129-PK-FL and P129-PK-d
43/44 showed significantly less IFN-α inhibitory effect than the parental P129 virus, the two Ingelvac attenuated strains, or the reference strain. In one example, P129-PK-FL or P
Co-infection with 129-PK-d43 / 44 virus resulted in an apparent enhancement of the IFN-α response to TGEv.

The results shown in Table 2 suggest the interferon-α inhibitory effect of wild-type PRRS viruses and derivatives adapted for growth in cell culture. The indicated PRRS virus stocks are grown on ZMAC-1 cells and the titer of these newly generated stocks is ZMAC
Determined by using -1 cells. Current IFN- in culture supernatants of porcine peripheral blood mononuclear cells exposed to the indicated PRRS virus stock for 18 hours with or without TGE virus
The amount of α was determined by ELISA. * Response to TGEv only.

The results shown in Table 3 show the interferon-α inhibitory effect of the genetically modified derivatives adapted for growth in wild type PRRS virus P129 and CD163-expressing PK-9 cells. PRRS virus stock (PBMC) indicated with or without TGE virus
The current amount of IFN-α in the culture supernatant of porcine peripheral blood mononuclear cells exposed to
Determined by ELISA. na = Not applicable: * Response to TGEv only.

Table 4 compares the wild type P129 virus and the PRRS Ingelvac vaccine:
The interferon-alpha inhibitory effect which P129-PK-FL and P129-PK-d43 / 44 virus attenuate | damps is shown. Current IFN-α in the culture supernatant of porcine peripheral blood mononuclear cells exposed to the indicated PRRS virus stock (PBMC) with or without TGE virus
The amount of was determined by ELISA. na = not applicable: * Responding to TGEv only.

The abundant amount of IFN-α secreted by PBMC in response to their exposure to TGEV is derived primarily from a subset of cells comprising less than 0.3% of the PBMC population. This rare but important cell subset is composed of plasmacytoid dendritic cells (PDC), which have accepted this name because of their characteristic plasmacytoid morphology. Furthermore P129-P
To examine the IFN-α phenotype of K-FL and P129-PK-d43 / 44 viruses, except that a freshly isolated PDC with> 95% purity was utilized,
A series of experiments were performed as described above. As shown in FIG. 1, this series of experiments confirmed that P129-PK-FL and P129-PK-d43 / 44 viruses cause negligible suppression of IFN-α induction by TGEV. Moreover, in one experiment, the apparent enhancement effect was P129-PK-FL and P129-PK-d43 / 44PR.
Observed by TGEV-mediated IFN-α induction by PDC in response to RS virus. In contrast, as shown in FIG. 1, Ingelvac PRRS MLV virus is
It showed a strong inhibitory effect on the N-α reaction.

The results described in the experimental section are P129-PK-FL and P129-PK-d4.
3/44 PRRS virus, as well as pCMV-S-P129-PK17-FL infectious c
We show that other derivatives of DNA clones have a greatly attenuated ability to suppress IFN-α induction by TGEV in infected PBMC or PDC cells. This includes wild type (low passage) PRRS virus and two commercially available modified live virus vaccines (Ingelvac PRRS MLV and Ingelvac PRRS ATP).
This is in marked contrast with the IFN inhibitory effect observed in The observation that the P129-PK-FL and P129-PK-d43 / 44 viruses are minimally suppressive at this important function of PDC is played when these cells mediate innate immunity against viral infection Given a major role, potentially significant.

Although the present invention provides a clinically effective commercial vaccine virus adapted to grow its recombinantly expressed CD163 receptor in permissive cells, such viruses and vaccines are historically “ It should also be noted that the "monkey cell" culture technology is not dependent on or developed in any way. See especially US Pat. No. 7,754,464.

  Example 6
Safety and efficacy of vaccine candidates

To evaluate their safety and efficacy against PRRS as vaccines, pCMV
-S-P129-PK17-FL infectious cDNA clones, P129-PK-FL (passage 7/24), P129-PK-d43 / 44 (passage 17/34), and P129-PK
-Three viruses derived from d43 / 44/46 (passage 17/34) were evaluated in a juvenile porcine respiratory disease model. The origin of these viruses is shown in FIG. 8 and the experimental design (administration group) is listed in Table 5. Low passage toxicity PRRSV isolate NADC20 is
Used for xenoantigen administration at 7 weeks of age (4 weeks after vaccination). Control dose groups include sham vaccine and commercial PRRS vaccine Ingelvac MLV.

The untreated (NT) group was as follows: The NT1 pig was a sentinel that monitored the health status of the causative pig. They were housed separately and necropsied prior to PRRSV challenge. NT2 pigs were contact controls housed separately in a cage between the two pups of the vaccinated pigs, a total of two per dose group for each T02 to T05. NT3 pigs were housed contact controls, one per vaccinated pig swine, for a total of two per treatment group (T01 to T05). Only NT3 pigs were assigned to the T01 group.

The rectal temperature of the vaccinated animals was measured after vaccination and compared to the T01 (sham vaccine) administration group. The result is shown in FIG. None of the vaccines caused fever. All groups averaged less than 104 ° C. throughout the observation period after vaccination.

The rectal temperature of pigs was measured after antigen administration. The result is shown in FIG. T01 pigs that had not been vaccinated showed sustained heat above 104 ° C. In contrast, 3
The type of vaccine significantly reduced fever after challenge. P129-PK-FL was most effective at lowering heat.

Animal weights were recorded before and after vaccination. The result is shown in FIG. Body weights were recorded on day -1 of the study (before vaccination), days 10, 24, 27 (prior to challenge) and 37 days. In pigs that have not been vaccinated, toxic NADC 20
Antigen challenge with virus almost completely eliminated weight gain during the 10 day observation period. The vaccine denied this effect to varying degrees.

The lungs of the challenged animals were examined at necropsy after challenge. The respective proportions of the lung containing lesions are shown in FIG. The T01 sham vaccine group had an average of 25.1% of pulmonary lesions. The vaccine attenuated lung lesions to varying degrees. P129-PK-FL attenuated pulmonary involvement to 1.1% and was most effective.

The severity of lung lesions was assessed using the Lung Assessment Score (LAS) as shown in FIG. Three vaccines attenuated LAS. P129-PK-FL attenuated the average LAS from 1.63 to 0.14 in the sham-vaccinated group.

Serum antibodies to PRRSV were caused by both vaccination and challenge. The ratio of IDEXX ELISA S / P was measured on days 27 and 37 of the study. The result is shown in FIG. Vaccination with P129-PK-FL induced the highest levels of anti-PRRS antibodies.

Viremia in the sera of pigs challenged was titrated on PAM cells. FIG. 7 shows the results (TCID ₅₀ / mL). P129-PK-FL was most effective in attenuating viremia after challenge.

While the invention has been described with reference to the above examples and accompanying documents, it is understood that the entire contents of each is incorporated by reference and includes various modifications and variations within the spirit and scope of the invention. Is done. Accordingly, the invention is limited only by the following claims.

Example 7
Induction of infectious CDNA CLONE PCMV-S-P129-PK17-FL from infectious CDNA clone PCMV-S-P129

  The PRRSV infectious cDNA clone of pCMV-S-P129-PK17-FL of the present invention can be obtained by using the technique of site-directed mutagenesis by one of ordinary skill in the art using the PRRSV infectious cDNA clone pCMV-S previously described. -It can be easily derived from P129. The PRRSV infectious cDNA clone pCMV-S-P129 is described in US Pat. No. 6,500,662 and is deposited at ATCC under accession number 203489. The DNA sequence of the PRRSV genome in this clone is also available in the Genbank (NCBI) database as accession number AF494042. Site-directed mutagenesis kits are commercially available from many suppliers and are capable of making many simultaneous nucleotide changes at multiple sites on a large plasmid. Such kits, Change-IT (TM) Multiple Mutation Site Directed Mutagenesis Kit (Affymetrix / USB), QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies-Stratagene Products), and AMAP Multi Site-directed Mutagenesis Kit (MBL International)), but is not limited to these.

  A list of nucleotide changes between the PRRSV infectious cDNA clone pCMV-S-P129 (available from ATCC) and the PRRSV infectious cDNA clone of the present invention pCMV-S-P129-PK17-FL is presented in Table 8. All changes are in the protein translation region of the genome. There are a total of 74 nucleotide changes, and using multiple time course reactions with 74 mutagenic primers and a commercial site-directed mutagenesis kit, the pCMV-S-P129 described herein is used. A plasmid molecule identical to the sequence to PK17-FL can be generated and introduced into the pCMV-S-P129 infectious clone. In practice, the cluster of mutations within about 50-60 nucleotides of each other can be altered using one mutagenic primer, so the same result can be obtained with fewer than 74 mutagenic primers. For example, using one mutagenic primer, nucleotides 735, 750, and 756 can be altered, as can nucleotides 965, 992, and 1009. Therefore, the number of primers was reduced to about 60.

  Of the 74 nucleotide changes, the majority (42) are synonymous or “silent”, meaning that they encode the same amino acid. These nucleotide changes are unlikely to have a measurable effect on interferon induction or viral repression phenotypes. The remaining 32 nucleotide changes are non-synonymous or “non-silent” and result in amino acid changes in the viral protein. These 32 nucleotide changes are predicted to be directly attributable to the viral interferon induction / suppression phenotype and are the same as shown by the virus encoded by the infectious clone pCMV-S-P129-PK17-FL. Should be changed to convert the virus encoded by the infectious clone pCMV-S-P129 against the interferon phenotype. Such changes require up to 32 mutagenic primers, but less when considering several clustering of related nucleotides.

  Example 8
De novo synthesis of infectious CDNA clone PCMV-S-P129-PK17-FL

As an alternative to site-directed mutagenesis, the PRRS viral genome of the present invention can be a chemically integrated de novo with appropriate 5 ′ and 3 ′ adapter sequences and a PRRS infectious cDNA clone It is cloned into pCMV-S-P129 (available from ATCC as accession number 203489) or similar plasmid backbone. Custom synthesis of genes longer than 50 kb (PRRSV genome is approximately 15.5 kb) is available as a commercial service from a number of vendors, GenScript, DNA2.0, and BioBasic Inc. Including, but not limited to. The synthetic viral genome is directed to the pCMV-S vector by replacing the viral genome of the infectious clone pCMV-S-P129 using 5 ′ PacI and 3 ′ SpeI restriction enzyme sites adjacent to the genome. To be cloned. To cleave the synthetic genome, a 24 nucleotide extension (5′-GCAGAGCTCG TTAATTAAA ACCGTC-genome-3 ′, including the underlined PacI site) was integrated at the 5 ′ end of the synthetic genome and an 83 nucleotide extension (5′-genome− AAAAAAAAAAAAAAAAAAAAAAATGCATATTTAAAATCCCAAGCCGAATTCCAGCACACTGGCGGCCGGTTACTAGTGAGCGGCCCGC-3 ′, including the underlined SpeI site) is incorporated at the 3 ′ end of the synthetic genome. After cleaving plasmids and synthetic genomes with PacI and SpeI, the appropriate fragments are purified using standard cloning techniques well known to those skilled in the art, joined using DNA-ligating enzymes, screened and propagated For E. coli.

Example 9
A vaccine consisting of P129-PKC12-FL virus is safe for use in 1-day-old pigs and is effective for at least 26 weeks.

Existing PRRS modified live vaccines are only encouraged for use in pigs over 2 weeks of age. Attenuated P129-PKC12-FL virus at passage 57 (2.13 log ₁₀ TCID _{50 in} 2 mL dose) is safe for use in 1-day-old neonatal pigs, and for 6 months after vaccination (26 A study was conducted to determine if it was sufficiently immunogenic to provide protection against toxic xenoantigen administration over a week). (As described in SEQ ID NO: 6, this is essentially the same as passage 52 virus).

A total of 22 out of 24 sows pregnant with PRRSV seronegative gave birth to healthy piglets. These pigs (pigs) were administered according to the assignment at about 1 day of age (Day 0) as a single 2.0 mL dose of sham vaccine or P129-PKC12-FL virus vaccine intramuscularly. . All healthy piglets in one litter / parturition frame were either the same sham vaccine (11 litters, 100 piglets) or P129-PKC12-FL virus vaccine (11 litters, 91 litters). Pig) was inoculated on the same day.
Sham vaccinated sows and piglets continued to show PRRSV negative throughout the vaccination period. No confounding disease factor was detected. The main variable used to demonstrate safety in piglets vaccinated at 1 day of age is clinical observation after vaccination. Serology was used to confirm successful vaccination of all piglets.

Clinical observations were observed and recorded for all piglets from day 1 to day 10 after vaccination. Of the 100 pigs that received the control product (T01) and the 91 piglets that received the test product (T02), 15 and 14 respectively were standard Not observed (see Table 11, table as numbered in this example). Piglets that were observed to be non-standard were further noted to have an abnormal general condition and / or depression.

The IDEX ELISA results showed that before vaccination all pigs were negative for PRRSV (S / P ratio <0.4) and all sham vaccine controls were serological during the vaccination phase of the study. Confirmed that he continued to show negative results. After vaccination, P
All pigs in the 129-PKC12-FL vaccine group were serologically positive by day 21 or 22 (S / P ratio> 0.4).

  This data is safe when attenuated P129-PKC12-FL virus at passage 57 is administered as a single 2 mL IM dose to piglets born from serologically negative sows at 1 day of age Is supported in cases where observed despite weaning at 21 or 22 days of age.

Prior to challenge, piglets vaccinated with sham vaccine and P129-PKC12-FL virus were re-contained in cages in the room (two piglets from the same vaccination group per cage) Each room would contain 12 sputum from each vaccination group. It was toxic heterologous PRRS isolate NADC20 that was challenged at 7, 18, or 26 weeks after vaccination. Equivalent NADC20 stock solution of antigen dose 4.0mL in _{2.27log 10 TCID 50 /mL(2.87log 10 TCID 50} / 4mL dose) (intramuscular injection nostril per 1.0mL and 2.0 mL) Met. NADC-20 is a highly virulent genotype 2 PRRS virus, Dr. of National Animal Disease Center USDA (Ames, Iowa). Supplied by KellyLager. The NADC-20ORF5 amino acid sequence is 94.5% identical to P129-PKC12-FL.

The main variable determining disease prevention was the proportion of lung lesions. Lung lesions were necropsied (1 after antigen administration)
0 days), and the percentage of cirrhosis of each leaf (left head, left middle, left tail, right head, right middle, right tail, and appendages) Scored and recorded as a percentage.

Seven weeks after vaccination, the percentage of lung lesions was increased in pigs that received sham vaccination.
It was significantly higher (P ≦ 0001) compared to piglets that received 129-PKC12-FL virus vaccination (Table 13).

At 18 weeks after vaccination, the rate of lung lesions was significantly higher in pigs that received sham vaccination compared to pigs that received P129-PKC12-FL virus vaccination (P ≦ 0001) (Table 14). ).

At 26 weeks after vaccination, the proportion of lung lesions was significantly higher in pigs that received sham vaccination compared to pigs that received P129-PKC12-FL virus vaccination (P ≦ 0001) (Table 15). .

  This result indicates that the attenuated vaccine virus P129-PKC12-FL is safe in 1-day-old piglets and can elicit strong immunological responses with an extraordinary period of immunity. A single dose protects 1-day-old piglets from toxic xenogeneic PRRS challenge for at least 26 weeks.

  These characteristics, such as safety in 1-day-old piglets and a 26-week period of PRRS immunity, are combined with multivalent combination porcine vaccines such as the bivalent PRRSV / Mycoplasma-Hyopneumoniae (M. hyo) vaccine, divalent PRRSV / pig. Circovirus type 2 (PCV2) vaccine, and trivalent PRRSV / M. The hyo / PCV2 vaccine is as beneficial as the monovalent PRRSV vaccine.

Example 10
A vaccine consisting of P129-PKC12-FL virus provides early expression of protective immunity.

Existing modified live PRRS vaccines are encouraged to be vaccinated at least 3-4 weeks prior to exposure to toxic PRRS strains. This time interval appears to be essential for establishing protective immunity. The studies described herein provide significant protection against toxic heterologous PRRS virus challenge delivered using the P129-PKC12-FL virus vaccine and two other commercial PRRS vaccines only 14 days after vaccination To demonstrate.
During the vaccination phase, groups of 18 pigs (3 weeks old) were housed in 4 separate rooms in 6 cages each. Pigs (pigs) were treated with sham vaccine (2 mL), attenuated P129-PKC12-FL passage 57 virus vaccine (3.62 log ₁₀ TCID ₅₀ during 2 mL administration), Ingelvac PRRS MLV vaccine, or Ingelvac PRRS ATP vaccine, single One intramuscular injection was administered at approximately 3 weeks of age (Day 0) according to manufacturer's instructions.

Prior to challenge, all remaining animals were recontained in 3 cages each with an empty cage between each containment cage, and multiple cages of animals from each administration group were placed in 4 rooms. Each was housed. It was the toxic heterologous PRRS isolate NADC20 (day 14) that was about 5 weeks old. The antigen dose was 2.07 log ₁₀ TCID _{50 /} mL (2.67 log ₁₀ T
It was comparable to NADC20 stock solution 4.0 mL (intramuscular injection nostril per 1.0mL plus 2.0 mL) at CID dose of _50/4 mL).

The main variable determining disease attenuation was the proportion of lung lesions in the vaccinated group versus the sham vaccine group. When compared to the sham vaccinated group, all vaccinated groups (P129-PKC12-FL, P = 0.0177; IngelvacPRRS ATP)
, P = 0.0255; Ingelvac PRRS MLV, P = 0.0137). When vaccinated groups were compared with each other, no significant difference was found (
Table 16).

  This result demonstrates that inducing partial immunity and disease attenuation by 14 days after vaccination can be a general property of modified live PRRS virus vaccines. This property is limited to the early acquired immunity (eg unique antibodies and cytotoxic T cells), the combination of innate immunity (eg induced interferon and natural killer cells) and / or between vaccine and challenge viruses. It may be caused by competition in pigs (pigs) for a large number of permissive host cells (eg alveolar macrophages). Regardless of the mechanism, this property protects pigs from diseases associated with natural or intentional PRRS infections (eg, intentional exposure to toxic PRRS endemic farm strains of upcoming young sows). It can be utilized.

  Thus, as described above, this property of early expression of PRRS immunity is due to the multivalent combination porcine vaccine, such as the bivalent PRRSV / Mycoplasma-Hyopneumoniae (M.hyo) vaccine, bivalent PRRSV / pig circovirus type 2 (PCV2). ) Vaccine, and trivalent PRRSV / M. The hyo / PCV2 vaccine is as beneficial as the monovalent PRRSV vaccine. A vaccine as described above that is beneficial in practicing the present invention (ie, providing early and safe vaccination when piglets are one day old, with selective development of immunity in the next two weeks) In the US Provisional Application No. 61/620189 by Niztel et al. Of the title “PCV / Mycoplasma hyopneumoniae / PRRSZ Combination Vaccine” filed on April 4, 2012 for all combination vaccine components. Reference is made to what is described, the complete disclosure of which is incorporated herein by reference as if set forth in its entirety.

  A unique PRRS vaccine that can be used in the practice of the present invention (ie, to provide early and safe vaccination when piglets are 1 day old with selective development of immunity in the next 2 weeks) (Or PRRS vaccine strain), see Murtaugh et al. , Vaccine, vol 29, pp. Attention is directed to Table 1 at 8192-8204, (2011), page 8196, including but not limited to Ingelvac PRRS MLV, Ingelvac PRRS ATP, and Subaxyn PRRS (derived from Iowa strain ISU-55). A number of the above viruses / vaccines are identified. It should be noted that vaccines that provide the above performance characteristics are also required to provide an immunization period of about 6 months.

Deposit of Biological Material The following biological material (see also US Pat. No. 6,500,662) can be found in 10801 University Blvd. , Manassas, Virginia, 20110-2209, USA, deposited with the American Cultured Cell Line Conservation Organization (ATCC) on November 19, 1998, and given the following accession number.

Plasmid pT7P129A, accession number 203488
Plasmid pCMV-S-P129, accession number 203489
The complete text and disclosure of the following US patents are hereby incorporated by reference as if set forth in full: US Pat. No. 6,500,662 and US Pat. No. 7,618,797.

Claims (6)

A vaccine for protecting pigs from infection by PRRS virus, comprising: (a) a polynucleotide molecule of SEQ ID NO: 6, or a solution of 0.5 M NaHPO ₄ , 7% SDS, 1 mM EDTA in said polynucleotide molecule North American PRRS encoded by any polynucleotide that hybridizes under high stringency conditions, including hybridization to filter-bound DNA at 65 ° C. and washing at 68 ° C. in 0.1 × SSC / 0.1% SDS A virus, (b) said encoding polynucleotide molecule, (c) said polynucleotide molecule in the form of a plasmid, or (d) a viral vector comprising said polynucleotide molecule, wherein PRRS virus has an effective immune protection reaction against PRRS virus infection It is possible to induce An amount effective to generate a carrier suitable for immune protection against infection and use in livestock, wherein the vaccine provides early and safe vaccination when piglets are one day old, A vaccine that supplies an immunization period of up to 6 months.   2. A vaccine according to claim 1, which is provided with the expression of immunity starting from 2 weeks after vaccination. A method of vaccinating pigs against infection with PRRS virus, comprising administering said vaccine between about 12 hours and 2 weeks of age, said vaccine encoding (a) North American PRRS virus An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule;
(B) an infectious RNA molecule encoding the North American PRRS virus;
(C) the polynucleotide molecule (a) in plasmid form, or (d) a viral vector comprising an infectious sequence.   4. The method of claim 3, wherein protective immunity occurs within about 14 days of vaccination.   The method of claim 4, wherein protective immunity occurs within about 28 days after vaccination at 15 days, 21 days after vaccination at 7 days, 21 days after vaccination at 1 day of age. .   4. The method of claim 3, wherein the period of protective immunity provided is up to 6 months.

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